Novel cancer-associated antigen

ABSTRACT

The present invention provides a novel cancer-associated antigen that can be used in the treatment and diagnosis of cancer. Further, the invention provides amino acid and nucleic acid sequence of the novel antigen, binding proteins, and immunoconjugates. The invention also relates to diagnostic and therapeutic methods and kits.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/097,336 filed Nov. 12, 2008, which is a national phase entryapplication of PCT/CA2006/002101 filed Dec. 21, 2006 (which designatedthe U.S.) which claims the benefit of U.S. provisional application Ser.No. 60/751,965 filed Dec. 21, 2005 (now abandoned). All of the priorapplications are incorporated herein in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“10241-232_SequenceListing.txt” (18,407 bytes), submitted via EFS-WEBand created on Apr. 19, 2011, is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a novel antigen associated with cancer andmethods and compositions for treating and detecting cancer.

BACKGROUND OF THE INVENTION

In the year 2000, an estimated 22 million people were suffering fromcancer worldwide and 6.2 millions deaths were attributed to this classof diseases. Every year, there are over 10 million new cases and thisestimate is expected to grow by 50% over the next 15 years (WHO, WorldCancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC Press,Lyon, 2003). Current cancer treatments are limited to invasive surgery,radiation therapy and chemotherapy, all of which cause eitherpotentially severe side-effects, non-specific toxicity and/ortraumatizing changes to ones body image and/or quality of life. Cancercan become refractory to chemotherapy reducing further treatment optionsand likelihood of success. The prognosis for some cancer is worse thanfor others and some are almost always fatal. In addition, some cancerswith a relatively high treatment success rate remain major killers dueto their high incidence rates.

One of the causes for the inadequacy of current cancer treatments istheir lack of selectivity for affected tissues and cells. Surgicalresection always involves the removal of apparently normal tissue as a“safety margin” which can increase morbidity and risk of complications.It also always removes some of the healthy tissue that may beinterspersed with tumor cells and that could potentially maintain orrestore the function of the affected organ or tissue. Radiation andchemotherapy will kill or damage many normal cells due to theirnon-specific mode of action. This can result in serious side-effectssuch as severe nausea, weight loss and reduced stamina, loss of hairetc., as well as increasing the risk of developing secondary cancerlater in life. Treatment with greater selectivity for cancer cells wouldleave normal cells unharmed thus improving outcome, side-effect profileand quality of life.

The selectivity of cancer treatment can be improved by targetingmolecules that are specific to cancer cells and not found on normalcells. These molecules can then be used as a target to antibody-baseddiagnostic or therapeutics or for drugs capable of altering theirfunction.

What little is known about the wild type Scratch protein, has beenobtained on the basis of conceptual translation and analysis of theresulting hypothetical protein sequence. Expression of Mammalian Scratch(Scrt) mRNA has been found confined to the brain, spinal cord and newlydifferentiating, postmitotic neurons suggesting a potential role inneuronal differentiation. The human mammalian Scratch gene has beenmapped to q24.3 (chromosome 8) Nakakura et al 2001a, PNAS vol 98 p4010-4015 and Nakakura et al 2001. Mol. Brain. Res. Vol 95 p 162-166.

Mammalian Scratch shares a SNAG domain with other zinc finger proteins,such as SNAI1, SNAI2, SNAI3, GFII and GFIIB. While quite a few labsworking on SNAG domains (Bathe E et al. 2000. Nat. Cell Biol, Vol.2:84-89; Kataoka H et al., 2000. Nucleic Acids Res. Vol. 28:626-633;Grimes H L et al. 1996. Mol. Cell. Biol. Vol. 16:6263-6272; Hemavathy Ket al. 2000. Mol. Cell. Biol. Vol: 20:5087-5095) and snail locomotorfunctionality have come across the over-expression of the Scrt gene, thepresence of the protein itself has not been shown thus far. Based on thehypothetical protein sequence, the Scratch protein should have five zincfinger domains and a SNAG domain responsible for a function intranscription repression. The sequence indicates that the resultingprotein would be an intra-nuclear one and in fact expression ofrecombinant Mammalian Scratch has been found confined to nucleus oftransfected cells (Nakakura et al 2001a, PNAS vol 98 p 4010-4015).

SUMMARY OF THE INVENTION

The present inventors have identified a novel cancer-associated protein.Accordingly, the invention provides a novel cancer-associated antigenthat can be used in the treatment and diagnosis of cancer. Inparticular, the antigen is associated with glioblastoma, melanoma,breast cancer, lung cancer, ovarian cancer, lymphoma, colon cancer,gastric cancers and/or prostate cancer.

The novel antigen is a variant of Mammalian Scratch. The variant has atransmembrane domain that is absent in wild type Scratch and as a resultthe protein of the invention is detectable on the cell surface.Accordingly, the invention includes an isolated protein comprising, acancer-associated variant of Mammalian Scratch that is expressed on thesurface of cancer cells. In an embodiment of the invention, thecancer-associated variant of Mammalian Scratch comprises the amino acidsequence defined by SEQ ID NO:1 or a variant thereof, or the amino acidsequence defined by SEQ ID NO:2 or a variant thereof.

Another aspect of the invention is an isolated protein comprising theamino acid sequence of SEQ ID NO:1 or a variant thereof or the aminoacid sequence of SEQ ID NO:2 or a variant thereof.

The invention also includes isolated nucleic acid sequences encoding theisolated protein of the invention, recombinant expression vectorscomprising the nucleic acid sequences of the invention and host cellscomprising the recombinant expression vectors of the invention.

In another aspect of the invention, the invention includes a method ofdetecting or monitoring cancer in a subject having or suspected ofhaving cancer, comprising detecting the isolated protein of theinvention on a cell in the sample, wherein cancer is indicated, if theisolated protein is detected on the cell.

In addition, the invention includes methods of detecting or monitoringcancer in a subject having or suspected of having cancer, comprisingdetecting the expression of the cancer-associated variant of MammalianScratch in the cell in the sample, wherein cancer is indicated, if theexpression of the cancer-associated variant of Mammalian Scratch isdetected in the cell.

A further aspect of the invention is a method of treating or preventingcancer in a subject by modulating the function or expression of aMammalian Scratch in the cancer cell.

The invention also includes pharmaceutical compositions comprising aneffective amount of the isolated proteins of the invention, the isolatednucleic acid sequences of the invention and/or the recombinantexpression vectors of the invention.

A further aspect of the invention is the use of the isolated proteins ofthe invention, the isolated nucleic acid sequences of the inventionand/or the recombinant expression vectors of the invention to elicit animmune response in a subject.

Another aspect of the invention is the use of the isolated proteins ofthe invention, the isolated nucleic acid sequences of the inventionand/or the recombinant expression vectors of the invention to treat orprevent cancer.

In addition, the invention includes methods for treating or preventingcancer in a subject comprising administering to the subject or a cellfrom the subject an effective amount of the isolated proteins of theinvention, the isolated nucleic acid sequences of the invention and/orthe recombinant expression vectors of the invention.

The invention also includes methods for inducing an immune response in asubject against the isolated protein of the invention comprisingadministering to the subject or a cell from the subject an effectiveamount of the isolated proteins of the invention, the isolated nucleicacid sequences of the invention and/or the recombinant expressionvectors of the invention.

A further aspect of the invention is a method of detecting or monitoringcancer in a subject, comprising the steps of:

-   -   (1) contacting a test sample taken from said subject with a        binding protein that binds specifically to an antigen on the        cancer cell to produce a binding protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows the glycan structures involved in the binding of VB3-011 tothe protein of the invention. Chondroitin sulphate A, also known asChondroitin-4-sulphate, (due to the presence of the Sulfate molecule atposition 4), is a linear molecule of repeating D-galactosamine andglucuronic acid (A). When two such CSA molecules get cross-linked via a2-6 alpha linkage, the glycan unit now represents the one recognized byHeamagglutinin (HA) (B).

FIG. 2 is a schematic representation of HA reagent immobilization. Atthe first level, the specificity of HA is enhanced by blocking theanti-HA/protein-G-sepharose epitopes. At the second level, it isimmobilized with protein-G-sepharose, simultaneously blocking anynon-specificity arising due to the anti-IgG coupling step. Level 3,reaction with ethanolamine, ensures that apart from the HA epitope, allother reactive amine groups are blocked, thus increasing specificity forHA.

FIG. 3 shows the results of a lectin-based purification of the proteinof the invention that is detected by VB3-011. Con-A and WGA lectinspulled down non-specific proteins, whereas HA pulled down only oneprotein present in the positive and absent in the negative cell line(FIG. 3A). U87MG, U118MG and A375 show a single band when purified withHA, whereas Panc-1 and Daudi show no detectable bands (FIG. 3B).

FIG. 4 shows the disappearance of the glycan residue due to degradationat room temperature. A 50 kDa band usually observed on IP with HAreagent, when let to sit at room temperature for an hour prior toseparation on SDS-PAGE, resulted in the degradation of the glycanresidue, thus showing the presence of a protein band devoid of theglycan portion of the antigen at 36 kDa.

FIG. 5 shows the presence of one single protein spot in the purifiedantigen complex, at Mw—36 kDa and pI=9.7. This represents a Western blotprofile of the 2D-PAGE obtained on VB3-011 antigen purification. Thecorresponding spot from the gel was used for ID purposes.

FIG. 6 and SEQ ID NO:3 show the complete mapping of the peptidesobtained and the sequence coverage of the wild type Mammalian Scratchmolecule, Accession # gi|13775236. The underlined amino acids representthe sequences of amino acids identified from MS analysis.

FIG. 7 and SEQ ID NO:4 show the sequence coverage obtained forgi|15928387 from MDA-MB-435S and a BLAST sequence comparison for435S-derived sequence and Scrt. MDA-MD-435S shows the presence of atruncated version of Scratch, i.e., 17.823 kDa protein gi|15928387, with100% homology to sequences 185-366 of SEQ ID NO:3 of the Wild typeMammalian Scratch molecule.

FIG. 8 shows the TOF-MS scans of peptides obtained from A-375 cell line,to detect the presence of all peptide ions in the sample. One hundredscans at 1200-1400V in the range of 100-1200 amu on a static nanosprayresulted in the recovery of a significant number of peptides, which whenanalyzed yielded a protein ID as Mammalian scratch. FIG. 8A representsthe TOF-MS scan with all multiply charged peptide ions and FIG. 8Brepresents the deconvoluted spectrum with singly charged peptide ions.

FIG. 9 shows TOF-MS scans of peptides obtained from U87MG cell line, todetect the presence of all peptide ions in the sample. Three hundredscans at 1200-1400V in the range of 100-1200 amu on a static nanosprayresulted in the recovery of a significant number of peptides, which whenanalyzed yielded a protein ID as Mammalian Scratch. FIG. 9A representsthe TOF-MS scan with all multiply charged peptide ions and FIG. 9Brepresents the deconvoluted spectrum with singly charged peptide ions.

FIG. 10 shows TOF-MS scans of peptides obtained from U118MG cell line,to detect the presence of all peptide ions in the sample. Twenty-sevenscans at 1200-1400V in the range of 100-1200 amu on a static nanosprayresulted in the recovery of a significant number of peptides, which whenanalyzed yielded a protein ID as Mammalian Scratch. FIG. 10A representsthe TOF-MS scan with all multiply charged peptide ions and FIG. 10Brepresents the deconvoluted spectrum with singly charged peptide ions.

FIG. 11 and SEQ ID NO:1 show the sequence coverage of peptides recoveredfrom mass spectrometry analysis as listed in Table 2. A total of 18peptides were recovered from in-gel tryptic digestion and 67% coverageof the protein was obtained. Underlined sequences represent the peptidesequences recovered. The highlighted peptide includes novel sequences.Specifically, the sequences in bold are the novel sequences and the onesin italics represent exact matches with Mammalian Scratch.

FIG. 12 shows the peptide mass fingerprinting results for the peptidesrecovered from VB3-011Ag. Protein scores greater than 77 were consideredsignificant. The only significant protein IDs observed pointed to theone antigen, known as Mammalian Scratch with a score of 149.

FIG. 13 shows that the identified antigen, Mammalian Scratch, has asignificant score of 149. Due to the nature of the database server andthe similarity/homology linked proteins, all the isoforms of thisprotein were pulled down as hits. MS/MS fragmentation and identity ofpeptides confirms that the antigen is Mammalian Scratch.

FIG. 14 shows the MS/MS ion fragmentation of the neutral peptide Mr.2402.978172, appearing as a triply charged molecule (802.00000, 3+). Thepeptide sequence (residues 92-117 of SEQ ID NO:3) exactly matched thepeptide from Scratch.

FIG. 15 shows the MS/MS ion fragmentation of the neutral peptide Mr.2134.985448, appearing as a doubly charged molecule (1068.500000, 2+).The flanking regions of the recovered peptide exactly matched thepeptide from Scratch; however the rest of the sequence showed not morethan 40% homology in the sequence information. The peptide sequence isresidues 2-23 of SEQ ID NO:2.

FIG. 16 shows representative photographs of VB3-011 immunohistochemicalstaining of neuroblastoma tissue (A-C) and melanoma tissue (D-F). Tissuesections are, (A)—Early stage neuroblastoma (Stage I, II, III non-N-mycamplified), 3+; (B)—Non-N-myc amplified Stage IV neuroblastoma, 2+;(C)—N-myc amplified Stage IV neuroblastoma, 3+. (D)—Early stage melanoma(Stage I-III), 3+; (E)—Stage IV melanoma, 3+; (F)-metastatic disease,3+. All photographs are shown at 400× magnification.

FIG. 17 and SEQ ID NOS:5 and 3 show a restriction map of Scratch-1.

FIG. 18 shows the in vitro cytotoxicity of VB6-011 in an MTS assay ofVB6-011 with antigen-positive cells MB-435S (open circle) andantigen-negative cells Panc-1 (black circle). Cells seeded at 1000 cellsper well, were incubated with the Fab-de-bouganin purified proteins.After 5 days incubation, the cell viability was measured and IC₅₀ wasdetermined.

DETAILED DESCRIPTION OF THE INVENTION (A) Definitions

The term “a cell” includes a single cell as well as a plurality orpopulation of cells. Administering an agent (such as a cancer-associatedprotein) to a cell includes both in vitro and in vivo administrations.

The term “administered systemically” as used herein means that theimmunoconjugate and/or other cancer therapeutic may be administeredsystemically in a convenient manner such as by injection (subcutaneous,intravenous, intramuscular, etc.), oral administration, inhalation,transdermal administration or topical application (such as topical creamor ointment, etc.), suppository applications, or means of an implant. Animplant can be of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.Suppositories generally contain active ingredients in the range of 0.5%to 10% by weight.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies. The antibodymay be from recombinant sources and/or produced in transgenic animals.The term “antibody fragment” as used herein is intended to include Fab,Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof and bispecific antibody fragments. Antibodies can befragmented using conventional techniques. For example, F(ab′)₂ fragmentscan be generated by treating the antibody with pepsin. The resultingF(ab′)₂ fragment can be treated to reduce disulfide bridges to produceFab′ fragments. Papain digestion can lead to the formation of Fabfragments. Fab, Fab′ and F(ab′)₂, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, bispecific antibody fragments and other fragmentscan also be synthesized by recombinant techniques.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm −5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “binding protein” as used herein refers to proteins thatspecifically bind to another substance such as a cancer-associatedantigen of the invention. In an embodiment, binding proteins areantibodies or antibody fragments.

By “biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects.

The terms “cancer-associated variant of Mammalian Scratch”,“cancer-associated antigen of the invention”, “tumor-associated antigenof the invention” or “isolated protein of the invention” as used hereinrefer to a novel variant of Mammalian Scratch that is expressed on thesurface of cancer cells or a variant thereof that is also expressed onthe surface of cancer cells. In one embodiment, the novelcancer-associated antigen has at least one transmembrane domain. Inspecific embodiments, the cancer-associated antigen of Mammalian Scratchis an isolated protein comprising the amino acid sequence defined by SEQID NO:1 or an isolated protein comprising the amino acid sequencedefined by SEQ ID NO:2.

The term “cancer cell” includes cancer or tumor-forming cells,transformed cells or a cell that is susceptible to becoming a cancer ortumor-forming cell.

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties.

The term “control” as used herein refers to a sample from a subject or agroup of subjects who are either known as having cancer or not havingcancer.

The term “controlled release system” as used means the immunoconjugateand/or other cancer therapeutic of the invention can be administered ina controlled fashion. For example, a micropump may deliver controlleddoses directly into the area of the tumor, thereby finely regulating thetiming and concentration of the pharmaceutical composition (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, vol. 2,pp. 115-138).

The term “derivative of a peptide” refers to a peptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The phrase “detecting or monitoring cancer” refers to a method orprocess of determining if a subject has or does not have cancer, theextent of cancer, the severity of cancer and/or grade of cancer.

The term “direct administration” as used herein means the cancertherapeutic may be administered, without limitation, intratumorally,intravascularly, and peritumorally. For example, the cancer therapeuticmay be administered by one or more direct injections into the tumor, bycontinuous or discontinuous perfusion into the tumor, by introduction ofa reservoir of the cancer therapeutic, by introduction of a slow-releaseapparatus into the tumor, by introduction of a slow-release formulationinto the tumor, and/or by direct application onto the tumor. By the modeof administration “into the tumor,” introduction of the cancertherapeutic to the area of the tumor, or into a blood vessel orlymphatic vessel that substantially directly flows into the area of thetumor, is included.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. Effective amounts of therapeutic may vary according to factorssuch as the disease state, age, sex, weight of the animal. Dosage regimemay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The term “eliciting an immune response” or “inducing an immune response”as used herein means initiating, triggering, causing, enhancing,improving or augmenting any response of the immune system, for example,of either a humoral or cell-mediate nature. The initiation orenhancement of an immune response can be assessed using assays known tothose skilled in the art including, but not limited to, antibody assays(for example ELISA assays), antigen specific cytotoxicity assays and theproduction of cytokines (for example ELISPOT assays). Preferably, theisolated proteins, nucleic acid sequences or recombinant expressionvectors of the present invention, and the method of the presentinvention, trigger or enhance a cellular immune response, morepreferably a T cell response.

The term “VB3-011 antibody” as used herein refers to an antibody withthe variable region of the antibody disclosed in WO 97/044461 which hasbeen shown to specifically bind to a variety of cancer cells and doesnot significantly bind to normal tissue or cells.

The term “isolated nucleic acid sequences” as used herein refers to anucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An isolated nucleic acid isalso substantially free of sequences which naturally flank the nucleicacid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid)from which the nucleic acid is derived. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded.

The term “isolated proteins” refers to a protein substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. It includes the novel cancer-associated antigen of theinvention.

“Mammalian Scratch” (gi|13775236; gi|46397014; gi|13129535) is a proteinencoded by a gene that has been mapped to q24.3 of human chromosome 8.From the analysis of the hypothetical protein sequence based onconceptual translation, mammalian scratch has 5 zinc finger domains anda SNAG domain. It is thought to be an intranuclear protein. Thehypothetical protein sequence is shown in SEQ ID NO:3.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentinvention may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The term “sample” as used herein refers to any fluid, cell or tissuesample from a subject which can be assayed for cancer.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences. In order todetermine the percentage of identity between two polypeptide sequences,the amino acid sequences of such two sequences are aligned, preferablyusing the Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J,1994, Nucleic Acids Res. 22 (22): 4673-4680), together with BLOSUM 62scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc. Natl. Acad.Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and gapextension penalty of 0.1, so that the highest order match is obtainedbetween two sequences wherein at least 50% of the total length of one ofthe sequences is involved in the alignment. Other methods that may beused to align sequences are the alignment method of Needleman and Wunsch(J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv.Appl. Math., 1981, 2: 482) so that the highest order match is obtainedbetween the two sequences and the number of identical amino acids isdetermined between the two sequences. Other methods to calculate thepercentage identity between two amino acid sequences are generally artrecognized and include, for example, those described by Carillo andLipton (SIAM J. Applied Math., 1988, 48:1073) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects. Generally,computer programs will be employed for such calculations. Computerprograms that may be used in this regard include, but are not limitedto, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP,BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215: 403).

The term “subject” as used herein refers to any member of the animalkingdom, preferably a mammal, more preferably a human being. In apreferred embodiment, the subject is suspected of having or has cancer.

As used herein, the phrase “treating or preventing cancer” refers toinhibiting of cancer cell replication, preventing transformation of acell to a cancer-forming cell, inhibiting of cancer spread (metastasis),inhibiting of tumor growth, reducing cancer cell number or tumor growth,decreasing in the malignant grade of a cancer (e.g., increaseddifferentiation), or improving cancer-related symptoms.

The term “variant” as used herein includes modifications or chemicalequivalents of the amino acid and nucleotide sequences of the presentinvention that perform substantially the same function as the proteinsor nucleic acid molecules of the invention in substantially the sameway. For example, variants of proteins of the invention include, withoutlimitation, conservative amino acid substitutions. Variants of proteinsof the invention also include additions and deletions to the proteins ofthe invention. In addition, variant peptides and variant nucleotidesequences include analogs and derivatives thereof. A variant of thecancer-associated antigen of the invention means a protein sequence thatis expressed on cancer cells but not normal cells.

(B) Novel Cancer-Associated Antigen

The invention provides a novel cancer-associated antigen that isexpressed on the surface of cancer cells and is not significantlyexpressed on the surface of normal cells. The novel cancer-associatedantigen is a variant of Mammalian Scratch. It has a transmembrane domainthat is not present in Mammalian Scratch. A sequence of thetransmembrane domain is shown in SEQ ID NO:2. A sequence of the cancerassociated variant is shown in SEQ ID NO:1.

In one embodiment, the invention provides an isolated protein comprisingthe amino acid sequence defined by SEQ ID NO:1 or a variant thereof. Inanother embodiment, the invention provides an isolated proteincomprising the amino acid sequence defined by SEQ ID NO:2. or a variantthereof.

The novel cancer-associated antigen is a variant of Mammalian Scratchthat is expressed on the surface of cancer cells. Accordingly, theinvention provides an isolated protein comprising a cancer-associatedvariant of Mammalian Scratch, wherein the cancer-associated variant ofMammalian Scratch is expressed on the surface of cancer cells. In oneembodiment, the cancer-associated variant of Mammalian Scratch comprisesthe amino acid sequence defined by SEQ ID NO:1. In another embodiment,the cancer-associated variant of Mammalian Scratch comprises the aminoacid sequence defined by SEQ ID NO:2.

A person skilled in the art will appreciate that the invention includesvariants to the amino acid sequences of SEQ ID NOS:1-2 wherein suchvariants are also cancer-associated antigens. Variants include chemicalequivalents to the sequences disclosed by the present invention. Suchequivalents include proteins that perform substantially the samefunction as the specific proteins disclosed herein in substantially thesame way. For example, equivalents include, without limitation,conservative amino acid substitutions.

In one embodiment, the variant amino acid sequences of the isolatedproteins of the invention have at least 50%, preferably at least 60%,more preferably at least 70%, most preferably at least 80%, and evenmore preferably at least 90% sequence identity to SEQ ID NOS:1 or 2.

The invention also provides an isolated nucleic acid sequence encodingthe isolated proteins of the invention. In one embodiment, the isolatednucleic acid has the sequence shown in SEQ ID NO:6. In addition, theinvention includes variants to the isolated nucleic acid sequences thatencode the isolated proteins of the invention. For example, the variantsinclude nucleotide sequences that hybridize to the nucleic acidsequences encoding the isolated proteins of the invention under at leastmoderately stringent hybridization conditions. The variant nucleic acidsequences will encode a protein that is a cancer-associated antigen.

The invention includes the use of the isolated proteins orcancer-associated antigens and corresponding nucleic acid sequences Forexample, the use of the isolated proteins of the invention to generatebinding proteins and immunoconjugates that can be used to treat orprevent cancer or that can be used to detect or monitor cancer in asubject. Accordingly, the invention includes the use of the isolatedproteins and nucleic acid sequences of the invention to treat or preventcancer and in the manufacture of a medicament to treat or prevent canceror for the diagnosis of cancer.

(C) Pharmaceutical Compositions, Methods and Uses of the NovelCancer-Associated Antigen

The invention provides a novel cancer-associated antigen that isexpressed on the surface of cancer cells and not significantly expressedon the surface of normal cells. Thus, the novel cancer-associatedantigen can be used in therapies to treat and prevent cancer, includingusing the isolated proteins of the invention to elicit an immuneresponse in vivo. In addition, the invention includes diagnostic methodsfor cancer that comprise detecting the novel cancer-associated antigen.

The cancer can be any cancer that expresses the cancer-associatedantigen of the invention on its cell surface. In one embodiment of theinvention, cancer includes, without limitation, stomach cancer, coloncancer, prostate cancer as well as cervical cancer, uterine cancer,ovarian cancer, pancreatic cancer, kidney cancer, liver cancer, head andneck cancer, squamous cell carcinoma, gastrointestinal cancer, breastcancer (such as carcinoma, ductal, lobular, and nipple), lung cancer,non-Hodgkin's lymphoma, multiple myeloma, leukemia (such as acutelymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenousleukemia, and chronic myelogenous leukemia), brain cancer,neuroblastoma, sarcomas, rectum cancer, bladder cancer, pancreaticcancer, endometrial cancer, plasmacytoma, lymphoma, and melanoma. In apreferred embodiment, the cancer includes, without limitation,glioblastoma, melanoma, breast cancer, lung cancer, ovarian cancer,lymphoma, colon cancer, gastric cancers and/or prostate cancer.

(i) Pharmaceutical Compositions

One aspect of the invention is a pharmaceutical composition comprisingan effective amount of the isolated protein of the invention inadmixture with a suitable diluent or carrier. Another aspect of theinvention is a pharmaceutical a composition comprising an effectiveamount of the isolated nucleic acid of the invention in admixture with asuitable diluent or carrier. A further aspect of the invention is apharmaceutical composition comprising an effective amount of therecombinant expression vector of the invention in admixture with asuitable diluent or carrier.

For example, the pharmaceutical compositions of the invention can beused to treat or prevent cancer. In addition, the pharmaceuticalcompositions can be used to elicit an immune response in a subjectagainst an isolated protein of the invention.

The compositions described herein can be prepared by known methods forthe preparation of pharmaceutically acceptable compositions that can beadministered to subjects, such that an effective quantity of the activesubstance is combined in a mixture with a pharmaceutically acceptablevehicle. Suitable vehicles are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20^(th)ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this basis,the compositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

Immunogenicity can be significantly improved if the immunizing agents(i.e. the isolated protein of the invention, and/or nucleic acidsequences coding therefore, and/or recombinant expression vectors)and/or composition is, regardless of administration format, co-immunizedwith an adjuvant. Commonly, adjuvants are used as a 0.05 to 1.0 percentsolution in phosphate buffered saline. Adjuvants enhance theimmunogenicity of an immunogen but are not necessarily immunogenic in ofthemselves. Adjuvants may act by retaining the immunogen locally nearthe site of administration to produce a depot effect facilitating aslow, sustained release of immunogen to cells of the immune system.Adjuvants can also attract cells of the immune system to an immunogendepot and stimulate such cells to elicit immune response. As such,embodiments of this invention encompass pharmaceutical compositionsfurther comprising adjuvants.

Adjuvants have been used for many years to improve the host immuneresponses to, for example, vaccines. Intrinsic adjuvants (such aslipopolysaccharides) normally are the components of killed or attenuatedbacteria used as vaccines. Extrinsic adjuvants are immunomodulatorswhich are typically non-covalently linked to antigens and are formulatedto enhance the host immune responses. Thus, adjuvants have beenidentified that enhance the immune response to antigens deliveredparenterally. Some of these adjuvants are toxic, however, and can causeundesirable side-effects making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines. The efficacy of alum inincreasing antibody responses to diphtheria and tetanus toxoids is wellestablished. Notwithstanding, it does have limitations. For example,alum is ineffective for influenza vaccination and inconsistently elicitsa cell mediated immune response with other immunogens. The antibodieselicited by alum-adjuvanted antigens are mainly of the IgG1 isotype inthe mouse, which may not be optimal for protection by some vaccinalagents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto immunogens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria and mineral oil, Freund's complete adjuvant,bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In one aspect of this invention, adjuvants useful in any of theembodiments of the invention described herein are as follows. Adjuvantsfor parenteral immunization include aluminum compounds (such as aluminumhydroxide, aluminum phosphate, and aluminum hydroxy phosphate). Theantigen can be precipitated with, or adsorbed onto, the aluminumcompound according to standard protocols. Other adjuvants such as RIBI(ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions of the invention include, for example Ser-63-Lys,Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (suchas a bacterial monophosphoryl lipid A (MPLA) of various sources (e.g.,E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigellaflexneri, saponins, or polylactide glycolide (PLGA) microspheres) canalso be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

A subject may be immunized with a pharmaceutical composition comprisingan isolated protein of the invention, an isolated nucleic acid sequenceof the invention and/or a recombinant expression vector of the inventionby any conventional route as is known to one skilled in the art. Thismay include, for example, immunization via a mucosal (e.g., ocular,intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, orurinary tract) surface, via the parenteral (e.g., subcutaneous,intradermal, intramuscular, intravenous, or intraperitoneal) route orintranodally. Preferred routes depend upon the choice of the immunogenas will be apparent to one skilled in the art. The administration can beachieved in a single dose or repeated at intervals. The appropriatedosage depends on various parameters understood by skilled artisans suchas the immunogen itself (i.e. peptide vs. nucleic acid (and morespecifically type thereof)), the route of administration and thecondition of the animal to be vaccinated (weight, age and the like).

A person skilled in the art will appreciate that the pharmaceuticalcompositions can be formulated for administration to subjects in abiologically compatible form suitable for administration in vivo. Thesubstances may be administered to living organisms including humans, andanimals. Administration of a therapeutically active amount of thepharmaceutical compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a substance may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of therecombinant protein of the invention to elicit a desired response in theindividual. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The pharmaceutical composition of the invention may be administeredsystemically. The pharmaceutical preparation may be administereddirectly to the cancer site. Depending on the route of administration,the pharmaceutical composition may be coated in a material to protectthe composition from the action of enzymes, acids and other naturalconditions that may inactivate the compound.

In accordance with one aspect of the present invention, thepharmaceutical composition is delivered to the subject by directadministration. The invention contemplates the pharmaceuticalcomposition being administered in at least an amount sufficient toachieve the endpoint, and if necessary, comprises a pharmaceuticallyacceptable carrier.

In accordance with another aspect, the pharmaceutical composition may beadministered in vitro. For example, lymphocytes may be removed from asubject with cancer and stimulated in vitro with the composition andthen infused back into the subject.

The invention also provides methods for reducing the risk ofpost-surgical complications comprising administering an effective amountof the pharmaceutical composition of the invention before, during, orafter surgery to treat cancer.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The pharmaceutical compositions of theinvention may be supplied, for example but not by way of limitation, asa lyophilized powder which is reconstituted with sterile water or salineprior to administration to the subject.

Pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include essentially chemically inert and nontoxiccompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, water,saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),diolesyiphosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the subject.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In various embodiments of the invention, the pharmaceutical compositionis directly administered systemically or directly to the area of thetumor(s).

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer. The dosageand type of pharmaceutical composition to be administered will depend ona variety of factors which may be readily monitored in human subjects.Such factors include the etiology and severity (grade and stage) of thecancer.

Clinical outcomes of cancer treatments using the pharmaceuticalcompositions of the invention are readily discernable by one of skill inthe relevant art, such as a physician. For example, standard medicaltests to measure clinical markers of cancer may be strong indicators ofthe treatment's efficacy. Such tests may include, without limitation,physical examination, performance scales, disease markers, 12-lead ECG,tumor measurements, tissue biopsy, cytoscopy, cytology, longest diameterof tumor calculations, radiography, digital imaging of the tumor, vitalsigns, weight, recordation of adverse events, assessment of infectiousepisodes, assessment of concomitant medications, pain assessment, bloodor serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.Furthermore, synergistic effects of a combination therapy comprising thepharmaceutical compositions of the invention and another cancertherapeutic may be determined by comparative studies with patientsundergoing monotherapy.

Another embodiment of the invention is a kit for treating or preventingcancer comprising an effective amount of the pharmaceutical compositionof the invention, and directions for the use thereof to treat thecancer.

In the majority of approved anticancer therapies, the anticancer therapyis used in combination with other anticancer therapies. Accordingly, theinvention provides a method of preventing or treating cancer using thepharmaceutical compositions of the invention in combination with atleast one additional anticancer therapy. The other cancer therapy may beadministered prior to, overlapping with, concurrently, and/or afteradministration of the pharmaceutical composition of the invention. Whenadministered concurrently, the pharmaceutical composition of theinvention and the other cancer therapeutic may be administered in asingle formulation or in separate formulations, and if separately, thenoptionally, by different modes of administration. The combination of oneor more pharmaceutical compositions of the invention and one or moreother cancer therapies may synergistically act to combat the tumor orcancer. The other cancer therapies include, without limitation,radiation and other anticancer therapeutic agents. These other cancertherapeutics may include, without limitation,2,2′,2″trichlorotriethylamine, 6-azauridine, 6-diazo-5-oxo-L-norleucine,6-mercaptopurine, aceglarone, aclacinomycins actinomycin, altretamine,aminoglutethimide, amsacrine, anastrozole, ancitabine, angiogeninantisense oligonucleotide, anthramycin, azacitidine, azaserine,aziridine, batimastar, bcl-2 antisense oligonucleotide, benzodepa,bicalutamide, bisantrene, bleomycin, buserelin, busulfan, cactinomycin,calusterone, carboplatin, carboquone, caminomycin, carmofur, carmustine,carubicin, carzinophilin, chlorambucil, chlornaphazine, chlormadinoneacetate, chlorozotocin, chromomycins, cisplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,defosfamide, demecolcine, denopterin, detorubicin, diaziquone,docetaxel, doxifluridine, doxorubicin, droloxifene, dromostanolone,edatrexate, eflomithine, elliptinium acetate, emitefur, enocitabune,epirubicin, epitiostanol, esorubicin, estramustine, etoglucid,etoposide, fadrozole, fenretinide, floxuridine, fludarabine,fluorouracil, flutamide, folinic acid, formestane, fosfestrol,fotemustine, gallium nitrate, gemcitabine, goserelin, hexestrol,hydroxyurea, idarubicin, ifosfamide, improsulfan, interferon-alpha,interferon-beta, interferon-gamma, interleukin-2, L-asparaginase,lentinan, letrozole, leuprolide, lomustine, lonidamine, mannomustine,marcellomycin, mechlorethamine, mechlorethamine oxide hydrochloride,medroxyprogesterone, megestrol acetate, melengestrol, melphalan,menogaril, mepitiostane, methotrexate, meturedepa, miboplatin,miltefosine, mitobronitol, mitoguazone, mitolactol, mitomycins,mitotane, mitoxantrone, mopidamol, mycophenolic acid, nilutamide,nimustine, nitracine, nogalamycin, novembichin, olivomycins,oxaliplatin, paclitaxel, pentostatin, peplomycin, perfosfamide,phenamet, phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim,plicamycin, podophyllinic acid 2-ethyl-hydrazide, polyestradiolphosphate, porfimer sodium, porfiromycin, prednimustine, procabazine,propagermanium, PSK, pteropterin, puromycin, quelamycin, ranimustine,razoxane, rodorubicin, roquinimex, sizofican, sobuzoxane,spirogermanium, streptonigrin, streptozocin, tamoxifen, taxotere,tegafur, temozolomide, teniposide, tenuzonic acid, testolacone,thiamiprine, thioguanine, thiotepa, Tomudex, topotecan, toremifene,triaziquone, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin,trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa,urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosinearabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide),hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids(e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR,CPT-11), vincristine, vinca alkyloids such as vinblastine.)podophyllotoxin, epipodophyllotoxin, VP-16 (etoposide), cytochalasin B,gramicidin D, ethidium bromide, emetine, anthracyclines (e.g.,daunorubicin), doxorubicin liposomal, dihydroxyanthracindione,mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin,capecitabine, carboplain, chlorabusin, cyclarabine, dactinomycin,floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino,mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab,campath-1, straplozocin, tretinoin, VEGF antisense oligonucleotide,vindesine, and vinorelbine. Compositions comprising one or more cancertherapeutics (e.g., FLAG, CHOP) are also contemplated by the presentinvention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) andG-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, andprednisone. For a full listing of cancer therapeutics known in the art,see, e.g., the latest editions of The Merck Index and the Physician'sDesk Reference.

Pharmaceutical compositions for combination therapy may also include,without limitation, antibiotics (e.g., dactinomycin, bleomycin,mithramycin, anthramycin), asparaginase, Bacillus and Guerin, diphtheriatoxin, procaine, tetracaine, lidocaine, propranolol, anti-mitoticagents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor,platelet derived growth factor, tissue plasminogen activator,antihistaminic agents, anti-nausea agents, etc.

Indeed, administration of an effective amount of a pharmaceuticalcomposition of the invention to a patient in need of such treatment mayresult in reduced doses of another cancer therapeutic having clinicallysignificant efficacy. Such efficacy of the reduced dose of the othercancer therapeutic may not be observed absent administration with thepharmaceutical compositions of the invention. Accordingly, the presentinvention provides methods for treating a tumor or cancer comprisingadministering a reduced dose of one or more other cancer therapeutics.

Moreover, combination therapy comprising the pharmaceutical compositionof the invention to a patient in need of such treatment may permitrelatively short treatment times when compared to the duration or numberof cycles of standard treatment regimens. Accordingly, the presentinvention provides methods for treating a tumor or cancer comprisingadministering one or more other cancer therapeutics for relatively shortduration and/or in fewer treatment cycles.

Thus, in accordance with the present invention, combination therapiescomprising a pharmaceutical composition of the invention and anothercancer therapeutic may reduce toxicity (i.e., side effects) of theoverall cancer treatment. For example, reduced toxicity, when comparedto a monotherapy or another combination therapy, may be observed whendelivering a reduced dose of a pharmaceutical composition of theinvention and/or other cancer therapeutic, and/or when reducing theduration of a cycle (i.e., the period of a single administration or theperiod of a series of such administrations), and/or when reducing thenumber of cycles.

Accordingly, the invention provides a pharmaceutical composition of theinvention further comprising one or more additional anticancertherapeutic, optionally in a pharmaceutically acceptable carrier.

The present invention also provides a kit comprising an effective amountof a pharmaceutical composition of the invention, optionally, incombination with one or more other cancer therapeutic, together withinstructions for the use thereof to treat cancer.

As stated above, combination therapy with a pharmaceutical compositionof the invention may sensitize the cancer or tumor to administration ofan additional cancer therapeutic. Accordingly, the present inventioncontemplates combination therapies for preventing, treating, and/orpreventing recurrence of cancer comprising administering an effectiveamount of a pharmaceutical composition of the invention prior to,subsequently, or concurrently with a reduced dose of a cancertherapeutic. For example, initial treatment with a pharmaceuticalcomposition of the invention may increase the sensitivity of a cancer ortumor to subsequent challenge with a dose of cancer therapeutic. Thisdose is near, or below, the low range of standard dosages when thecancer therapeutic is administered alone, or in the absence of apharmaceutical composition of the invention. When concurrentlyadministered, the pharmaceutical composition of the invention may beadministered separately from the cancer therapeutic, and optionally, viaa different mode of administration.

In an alternate embodiment, administration of the additional cancertherapeutic may sensitize the cancer or tumor to pharmaceuticalcomposition of the invention. In such an embodiment, the additionalcancer therapeutic may be given prior to administration of apharmaceutical composition of the invention.

In one embodiment, the additional cancer therapeutic comprisescisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at a doseranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescarboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging fromapproximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescyclophosphamide, e.g., CYTOXAN (Bristol Myers Squibb), at a doseranging from approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to10, 11 to 20, or 21 to 40 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisescytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging fromapproximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to100 mg/m²/cycle. In another embodiment, the additional cancertherapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.),at a dose ranging from approximately 5 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose rangingfrom approximately 15 to 250 mg/m²/cycle or ranging from approximately0.2 to 2 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisestopotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging fromapproximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5mg/m²/Cycle. In another embodiment, the additional cancer therapeuticcomprises irinotecan, e.g., CAMPTOSAR (Pharmacia & Upjohn), at a doseranging from approximately 5 to 9, 10 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesfludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging fromapproximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to2000 mg/m²/cycle, 300 to 1000 mg/m²/cycle, 400 to 800 mg/m²/cycle, or500 to 700 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdocetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging fromapproximately 6 to 10, 11 to 30, or 31 to 60 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisespaclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging fromapproximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprises5-fluorouracil at a dose ranging from approximately 0.5 to 5mg/kg/cycle, 1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesdoxorubicin, e.g., ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX(Bristol Myers Squibb), at a dose ranging from approximately 2 to 4, 5to 8, 9 to 15, 16 to 30, or 31 to 60 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesetoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging fromapproximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging fromapproximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvincristine, e.g., ONCOVIN (Eli Lilly), at a dose ranging fromapproximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesmethotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6to 10, or 11 to 20 mg/m²/cycle.

In another embodiment, a pharmaceutical composition of the invention isadministered in combination with at least one other immunotherapeuticwhich includes, without limitation, rituxan, rituximab, campath-1,gemtuzumab, and trastuzutmab.

In another embodiment, a pharmaceutical composition of the invention isadministered in combination with one or more anti-angiogenic agentswhich include, without limitation, angiostatin, thalidomide, kringle 5,endostatin, Serpin (Serine Protease Inhibitor), anti-thrombin, 29 kDaN-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin,16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragmentof platelet factor-4, a 13 amino acid peptide corresponding to afragment of platelet factor-4 (Maione et al., 1991, Cancer Res.51:2077-2083), a 14-amino acid peptide corresponding to a fragment ofcollagen I (Tolsma et al., 1993, J. Cell Biol. 122:497-511), a 19 aminoacid peptide corresponding to a fragment of Thrombospondin I (Tolsma etal., 1993, J. Cell Biol. 122:497-511), a 20-amino acid peptidecorresponding to a fragment of SPARC (Sage et al., 1995, J. Cell.Biochem. 57:1329-1334), and a variant thereof, including apharmaceutically acceptable salt thereof.

In another embodiment, a pharmaceutical composition of the invention isadministered in combination with a regimen of radiation therapy. Thetherapy may also comprise surgery and/or chemotherapy. For example, apharmaceutical composition of the invention may be administered incombination with radiation therapy and cisplatin (Platinol),fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), and/orpaclitaxel (Taxol). Treatment with a pharmaceutical composition of theinvention may allow use of lower doses of radiation and/or less frequentradiation treatments, which may for example, reduce the incidence ofsevere sore throat that impedes swallowing function potentiallyresulting in undesired weight loss or dehydration.

In another embodiment, a pharmaceutical composition is administered incombination with one or more cytokines which include, withoutlimitation, a lymphokine, tumor necrosis factors, tumor necrosisfactor-like cytokine, lymphotoxin, interferon, macrophage inflammatoryprotein, granulocyte monocyte colony stimulating factor, interleukin(including, without limitation, interleukin-1, interleukin-2,interleukin-6, interleukin-12, interleukin-15, interleukin-18), and avariant thereof, including a pharmaceutically acceptable salt thereof.

In yet another embodiment, a pharmaceutical composition of the inventionis administered in combination with a cancer vaccine or biologicalagents including, without limitation, autologous cells or tissues,non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,Mycobacterial cell wall-DNA complexes, melanocyte lineage proteins, andmutated, tumor-specific antigens.

In yet another embodiment, a pharmaceutical composition is administeredin association with hormonal therapy. Hormonal therapeutics include,without limitation, a hormonal agonist, hormonal antagonist (e.g.,flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g.,dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen,testosterone, progestin).

In yet another embodiment, a pharmaceutical composition is administeredin association with a gene therapy program to treat or prevent cancer.

Combination therapy may thus increase the sensitivity of the cancer ortumor to the administered pharmaceutical composition of the inventionand/or additional cancer therapeutic. In this manner, shorter treatmentcycles may be possible thereby reducing toxic events. The cycle durationmay vary according to the specific cancer therapeutic in use. Theinvention also contemplates continuous or discontinuous administration,or daily doses divided into several partial administrations. Anappropriate cycle duration for a specific cancer therapeutic will beappreciated by the skilled artisan, and the invention contemplates thecontinued assessment of optimal treatment schedules for each cancertherapeutic. Specific guidelines for the skilled artisan are known inthe art. See, e.g., Therasse et al., 2000, “New guidelines to evaluatethe response to treatment in solid tumors. European Organization forResearch and Treatment of Cancer, National Cancer Institute of theUnited States, National Cancer Institute of Canada,” J Natl Cancer Inst.February 2; 92(3):205-16.

It is contemplated that a pharmaceutical composition of the inventionmay be administered by any suitable method such as injection, oraladministration, inhalation, transdermal or intratumorally, whereas anyother cancer therapeutic may be delivered to the patient by the same oranother mode of administration. Additionally, where multiple cancertherapeutics are intended to be delivered to a subject, a pharmaceuticalcomposition of the invention and one or more of the other cancertherapeutics may be delivered by one method, whereas other cancertherapeutics may be delivered by another mode of administration.

The invention also provides kits comprising an effective amount of apharmaceutical composition of the invention, optionally, in combinationwith one or more other cancer therapeutic agent, together withinstructions for the use thereof.

(ii) Diagnostic Methods

The novel cancer-associated antigen is expressed on cancer cells and isnot significantly expressed on normal cells, thus the detection of thenovel cancer-associated antigen can be used as a diagnostic method forcancer.

One embodiment of the invention is a method of detecting or monitoringcancer in a subject having or suspected of having cancer, comprisingdetecting a cancer-associated variant of Mammalian Scratch on a cell inthe sample, wherein cancer is indicated, if the cancer-associatedvariant of Mammalian Scratch is detected on the cell.

In an embodiment of the invention, a method is provided for detectingcancer cells in a subject comprising:

-   -   (a) providing a sample from the subject;    -   (b) detecting the level of the cancer-associated antigen in the        sample; and    -   (c) comparing the level of the cancer-associated antigen in the        sample to a control sample, wherein increased levels of the        cancer-associated antigen as compared to the control indicates        that the subject has cancer.

The phrase “detecting the level of the cancer-associated antigen”includes the detection of the levels of the cancer-associated antigen aswell as detection of the levels of nucleic acid molecules encoding thecancer-associated antigen. Examples of methods for detecting proteinsand nucleic acids are discussed in greater detail below.

The cancer-associated antigen preferably comprises the sequence shown inSEQ ID NO:2, more preferably, SEQ ID NO:1.

The term sample can be any sample containing cancer cells that onewishes to detect including, but not limited to, biological fluids(including blood, serum, ascites), tissue extracts, freshly harvestedcells, and lysates of cells which have been incubated in cell cultures.

The term “control sample” includes any sample that can be used toestablish a base or normal level, and may include tissue samples takenfrom healthy persons or samples mimicking physiological fluid. Thecontrol sample can also be a sample from the subject from another pointin time, e.g. prior to cancer therapy.

The method of the invention may be used in the diagnosis and staging ofthe cancer. The invention may also be used to monitor the progression ofa cancer and to monitor whether a particular treatment is effective ornot. In particular, the method can be used to confirm the absence orremoval of all tumor tissue following surgery, cancer chemotherapy,and/or radiation therapy. The methods can further be used to monitorcancer chemotherapy and tumor reappearance.

In an embodiment, the invention contemplates a method for monitoring theprogression of cancer in a subject, comprising:

-   -   (a) providing a sample from a subject;    -   (b) determining the level of the cancer-associated antigen        expression in the sample;    -   (c) repeating steps (a) and (b) at a later point in time and        comparing the result of step (b) with the result of step (c)        wherein a difference in the level of the cancer-associated        antigen expression is indicative of the progression of the        cancer in the subject.

In particular, increased levels of the cancer-associated antigen at thelater time point may indicate that the cancer is progressing and thatthe treatment (if applicable) is not being effective. In contrast,decreased levels of the cancer-associated antigen at the later timepoint may indicate that the cancer is regressing and that the treatment(if applicable) is effective.

A number of techniques can be used to detect the cancer-associatedvariant of Mammalian Scratch on a cell. For example, binding proteinssuch as antibodies that bind to the cancer-associated variant ofMammalian Scratch can be used in immunoassays to detect cell surfaceexpression of the cancer-associated variant of Mammalian Scratch. Aperson skilled in the art will appreciate that a number of techniquescan be used to detect and/or quantify cell surface expression of thecancer-associated variant of Mammalian Scratch, including, withoutlimitation, Western blots, immunoprecipitation followed by SDS-PAGE,immunocytochemistry, FACS, protein arrays, and the like.

Methods for Detecting Nucleic Acid Molecules

In one embodiment, the methods of the invention involve the detection ofnucleic acid molecules encoding the cancer-associated antigen. Thoseskilled in the art can construct nucleotide probes for use in thedetection of nucleic acid sequences encoding the cancer-associatedantigen in samples. Suitable probes can be prepared based on the nucleicacid sequence shown in SEQ ID NO:6 or SEQ ID NO:25. Suitable probesinclude nucleic acid molecules based on nucleic acid sequences encodingat least 5 sequential amino acids from regions of the cancer-associatedantigen, preferably they comprise 15 to 30 nucleotides. A nucleotideprobe may be labeled with a detectable substance such as a radioactivelabel which provides for an adequate signal and has sufficient half-lifesuch as ³²P, ³H, ¹⁴C or the like. Other detectable substances which maybe used include antigens that are recognized by a specific labeledantibody, fluorescent compounds, enzymes, antibodies specific for alabeled antigen, and luminescent compounds. An appropriate label may beselected having regard to the rate of hybridization and binding of theprobe to the nucleotide to be detected and the amount of nucleotideavailable for hybridization. Labeled probes may be hybridized to nucleicacids on solid supports such as nitrocellulose filters or nylonmembranes as generally described in Sambrook et al, 1989, MolecularCloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may beused to detect genes, preferably in human cells, that encode thecancer-associated antigen. The nucleotide probes may also be useful inthe diagnosis of disorders involving the cancer-associated antigen, inmonitoring the progression of such disorders, or in monitoring atherapeutic treatment. In an embodiment, the probes are used in thediagnosis of, and in monitoring the progression of cancer, preferablygynecological cancer.

The probe may be used in hybridization techniques to detect genes thatencode the cancer-associated antigen. The technique generally involvescontacting and incubating nucleic acids (e.g. recombinant DNA molecules,cloned genes) obtained from a sample from a subject or other cellularsource with a probe under conditions favorable for the specificannealing of the probes to complementary sequences in the nucleic acids.After incubation, the non-annealed nucleic acids are removed, and thepresence of nucleic acids that have hybridized to the probe if any aredetected.

The detection of nucleic acid molecules may involve the amplification ofspecific gene sequences using an amplification method such as polymerasechain reaction (PCR), followed by the analysis of the amplifiedmolecules using techniques known to those skilled in the art. Suitableprimers can be routinely designed by one of skill in the art.

Hybridization and amplification techniques described herein may be usedto assay qualitative and quantitative aspects of expression of genesencoding the cancer-associated antigen. For example, RNA may be isolatedfrom a cell type or tissue known to express a gene encoding thecancer-associated antigen, and tested utilizing the hybridization (e.g.standard Northern analyses) or PCR techniques which are known in theart.

The primers and probes may be used in the above described methods insitu i.e. directly on tissue sections (fixed and/or frozen) of subjecttissue obtained from biopsies or resections.

Accordingly, the present invention provides a method of detecting cancercells in a subject comprising:

-   -   (a) providing a sample from the subject;    -   (b) extracting nucleic acid molecules encoding the        cancer-associated antigen or portion thereof from the sample;    -   (c) amplifying the extracted nucleic acid molecules using the        polymerase chain reaction;    -   (d) determining the presence of nucleic acid molecules encoding        the cancer-associated antigen; and    -   (e) comparing the level of the nucleic acid molecules encoding        the cancer-associated antigen in the sample to a control sample,        wherein increased levels of the nucleic acid molecules encoding        the cancer-associated antigen as compared to the control        indicates that the subject has cancer.

In a preferred embodiment, the nucleic acid molecule encodes acancer-associated antigen that comprises SEQ ID NO:2, more preferablySEQ ID NO:1. In a specific embodiment, the nucleic acid moleculecomprises the sequence shown in SEQ ID NO:6 or a diagnostic fragmentthereof. In another embodiment, the nucleic acid molecule comprises thesequence shown in SEQ ID NO:25 (which encodes the transmembrane fragmentshown in SEQ ID NO:2) or a diagnostic fragment thereof.

The methods of the invention described herein may also be performedusing microarrays, such as oligonucleotide arrays, cDNA arrays, genomicDNA arrays, or tissue arrays. Preferably the arrays are tissuemicroarrays.

In a preferred example, an RNA expression product encoding thecancer-associated variant of Mammalian Scratch is used to detect theexpression of the cancer-associated variant of Mammalian Scratch by thecell. One skilled in the art will appreciate that the RNA expressionproduct can be detected or quantified by detecting mRNA encoding thecancer-associated variant of Mammalian Scratch or a fragment thereof, oroligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, syntheticRNA, or other combinations of naturally occurring or modifiednucleotides which specifically and/or selectively hybridize to the mRNAencoding the cancer-associated variant of Mammalian Scratch or afragment thereof.

A number of methods can be used to detect and/or quantify RNA expressionof the cancer-associated variant of Mammalian Scratch by a cellincluding RT-PCR, nuclease protection assays, such as ribonucleaseprotection assays and S1 nuclease assays, and Northern blots and thelike.

In a particular embodiment, the inventors have prepared PCR primers thatamplify both variant and wildtype scratch (SEQ ID NO:26) or only variantscratch (SEQ ID NO:27) as described in Example 4. Using such primersallows one to distinguish between variant and wild type scratch.

The inventors have also determined that the sequence of wild typeMammalian Scratch contains a KpnI restriction site at nucleotide 118that is not present in the cancer-associated variant. Therefore, to testif a cancer expresses the variant, the amplified PCR product can bedigested with the KpnI restriction enzyme followed by gelelectrophoresis. If the cells being tested express wildtype MammalianScratch then 2 fragments of 67 bp and 93 bp will be detected. If thecells express the cancer-associated variant then the size of the PCRproduct will be the same as the undigested control.

Accordingly, the present invention provides a method of detecting cancercells or monitoring cancer in a subject having or suspected of havingcancer comprising:

-   -   (a) providing a sample from the subject;    -   (b) extracting nucleic acid molecules encoding wild type scratch        or the cancer-associated variant of scratch from the sample;    -   (c) digesting the nucleic acid molecules with a KpnI restriction        enzyme; and    -   (d) determining the size of the digested nucleic acid molecules        wherein the presence of undigested nucleic acid molecules        indicates that the subject has cancer.

Methods for Detecting the Cancer-Associated Antigen

In another embodiment, the methods of the invention involve thedetection of the cancer-associated antigen. In one embodiment, thecancer-associated antigen is detected using antibodies that specificallybind to the cancer-associated antigen. Antibodies to thecancer-associated antigen may be prepared using techniques known in theart.

Antibodies specifically reactive with the cancer-associated antigen, orderivatives, such as enzyme conjugates or labeled derivatives, may beused to detect the cancer-associated antigen in various samples (e.g.biological materials). They may be used as diagnostic or prognosticreagents and they may be used to detect abnormalities in the level ofprotein expression, or abnormalities in the structure, and/or temporal,tissue, cellular, or subcellular location of the cancer-associatedantigen. In vitro immunoassays may also be used to assess or monitor theefficacy of particular therapies. The antibodies of the invention mayalso be used in vitro to determine the level of expression of a geneencoding the cancer-associated antigen in cells genetically engineeredto produce the cancer-associated antigen.

The antibodies may be used in any known immunoassays which rely on thebinding interaction between an antigenic determinant of thecancer-associated antigen and the antibodies. Examples of such assaysare radioimmunoassays, enzyme immunoassays (e.g. ELISA),immunofluorescence, immunoprecipitation, latex agglutination,hemagglutination, and histochemical tests. The antibodies may be used todetect and quantify the cancer-associated antigen in a sample in orderto determine its role in cancer and to diagnose the cancer.

In particular, the antibodies of the invention may be used inimmuno-histochemical analyses, for example, at the cellular andsubcellular level, to detect an the cancer-associated antigen, tolocalize it to particular cells and tissues, and to specific subcellularlocations, and to quantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect thecancer-associated antigen. Generally, an antibody of the invention maybe labeled with a detectable substance and the cancer-associated antigenmay be localised in tissues and cells based upon the presence of thedetectable substance. Examples of detectable substances include, but arenot limited to, the following: radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I,¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),luminescent labels such as luminol; enzymatic labels (e.g., horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase,acetylcholinesterase), biotinyl groups (which can be detected by markedavidin e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or calorimetric methods),predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). In some embodiments,labels are attached via spacer arms of various lengths to reducepotential steric hindrance. Antibodies may also be coupled to electrondense substances, such as ferritin or colloidal gold, which are readilyvisualised by electron microscopy.

The antibody or sample may be immobilized on a carrier or solid supportwhich is capable of immobilizing cells, antibodies etc. For example, thecarrier or support may be nitrocellulose, or glass, polyacrylamides,gabbros, and magnetite. The support material may have any possibleconfiguration including spherical (e.g. bead), cylindrical (e.g. insidesurface of a test tube or well, or the external surface of a rod), orflat (e.g. sheet, test strip). Indirect methods may also be employed inwhich the primary antigen-antibody reaction is amplified by theintroduction of a second antibody, having specificity for the antibodyreactive against the cancer-associated antigen. By way of example, ifthe antibody having specificity against the cancer-associated antigen isa rabbit IgG antibody, the second antibody may be goat anti-rabbitgamma-globulin labeled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, thecancer-associated antigen may be localized by radioautography. Theresults of radioautography may be quantitated by determining the densityof particles in the radioautographs by various optical methods, or bycounting the grains.

Labeled antibodies against the cancer-associated antigen may be used inlocating tumor tissue in subjects undergoing surgery i.e. in imaging.Typically for in vivo applications, antibodies are labeled withradioactive labels (e.g. iodine-123, iodine-125, iodine-131, gallium-67,technetium-99, and indium-111). Labeled antibody preparations may beadministered to a subject intravenously in an appropriate carrier at atime several hours to four days before the tissue is imaged. During thisperiod unbound fractions are cleared from the subject and the onlyremaining antibodies are those associated with tumor tissue. Thepresence of the isotope is detected using a suitable gamma camera. Thelabeled tissue can be correlated with known markers on the subject'sbody to pinpoint the location of the tumor for the surgeon.

Accordingly, in another embodiment the present invention provides amethod for detecting cancer in a subject comprising:

-   -   (a) providing a sample from the subject;    -   (b) contacting the sample with an antibody that binds to the        cancer-associated antigen;    -   (c) detecting the level of the cancer-associated antigen in the        sample; and    -   (d) comparing the level of the cancer-associated antigen in the        sample to a control sample, wherein increased levels of the        cancer-associated antigen as compared to the control indicates        that the subject has cancer.        (iii) Therapeutic Methods

As mentioned above, the novel cancer-associated antigen is present oncancer cells, but not significantly on normal cells. Thus, the novelcancer-associated antigen can be used in therapeutic methods to preventand treat cancer. In addition, the novel cancer-associated antigen orfragments thereof can be used to elicit an immune response in vivo, forexample in a vaccine, or in vitro.

One embodiment of the invention is the use of an isolated protein of theinvention or fragment thereof in the manufacture of a medicament totreat or prevent cancer. Yet another embodiment of the invention is theuse of an isolated protein of the invention or fragment thereof to treator prevent cancer. A further embodiment of the invention is the use ofan isolated protein of the invention or fragment thereof in themanufacture of a medicament to elicit an immune response. Yet anotherembodiment of the invention is the use of an isolated protein of theinvention or fragment thereof to elicit an immune response.

The invention also includes the use of an isolated nucleic acid sequenceof the invention in the manufacture of a medicament to treat or preventcancer. The invention further includes the use of an isolated nucleicacid sequence of the invention to treat or prevent cancer. In addition,the invention includes the use of an isolated nucleic acid sequence ofthe invention in the manufacture of a medicament to elicit an immuneresponse. The invention further includes the use of an isolated nucleicacid sequence of the invention to elicit an immune response.

A further embodiment of the invention is the use of the recombinantexpression vector of the invention in the manufacture of a medicament totreat or prevent cancer. Yet another embodiment of the invention is theuse of the recombinant expression vector of the invention to treat orprevent cancer. Also, the invention includes the use of the recombinantexpression vector of the invention in the manufacture of a medicament toelicit an immune response in a subject. Yet another embodiment of theinvention is the use of the recombinant expression vector of theinvention to elicit an immune response in a subject.

An additional embodiment of the invention is a method of treating orpreventing cancer comprising administering an effective amount of anisolated protein of the invention or a fragment thereof to a subject orcell in need thereof. In addition, the invention includes a method oftreating or preventing cancer comprising administering an effectiveamount of the isolated nucleic acid sequence of the invention to asubject or cell in need thereof. Further, the invention includes amethod of treating or preventing cancer comprising administering aneffective amount of the recombinant expression vector of the inventionto a subject or cell in need thereof.

Another embodiment of the invention is a method of inducing an immuneresponse in a subject against an isolated protein of the invention,comprising administering an effective amount of the isolated protein ofthe invention or a fragment thereof to a subject or cell in needthereof. In addition, the invention includes a method of inducing animmune response in a subject against the isolated protein of theinvention, comprising administering an effective amount of the isolatednucleic acid sequence of the invention to a subject or cell in needthereof. Further, the invention includes a method of inducing an immuneresponse in a subject against the isolated protein of the inventioncomprising administering an effective amount of the recombinantexpression vector of the invention to a subject or cell in need thereof.

The above methods include both in vivo and in vitro administration ofthe isolated protein of the invention. For in vitro uses, the proteincan be used to stimulate lymphocytes obtained from the patient which arethen re-infused into the subject to mount an immune response against thecancer cells expressing the cancer-associated antigen.

A further aspect of the invention is a method of treating or preventingcancer in a subject by modulating the activity or expression of thecancer-associated variant of Mammalian Scratch on or in a cancer cell.

In one embodiment of the invention, the method of treating or preventingcancer in a subject comprises preventing or decreasing the function ofthe cancer-associated variant of Mammalian. In one embodiment of theinvention, a binding protein of the invention is used to prevent ordecrease the function of the cancer-associated variant of MammalianScratch.

In another embodiment of the invention, the function of thecancer-associated variant of Mammalian Scratch is prevented or decreasedby decreasing or preventing the expression of the cancer-associatedvariant of Mammalian Scratch in the cell.

Standard techniques can be used to prevent or decrease the expression ofthe cancer-associated variant of Mammalian Scratch in a cell includingusing antisense, triple helix, or ribozyme molecules reactive to thetranscripts of the cancer-associated variant of Mammalian Scratch gene.

For example, standard techniques can be utilized for the production ofantisense nucleic acid molecules, i.e., molecules which arecomplementary to a sense nucleic acid encoding a polypeptide ofinterest, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire coding strand,or to only a portion thereof, e.g., all or part of the protein codingregion (or open reading frame). An antisense nucleic acid molecule canbe antisense to all or part of a non-coding region of the coding strandof a nucleotide sequence encoding a polypeptide of interest. Thenon-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′sequences that flank the coding region and are not translated into aminoacids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

Antisense nucleic acid molecules administered to a subject or generatedin situ such that they hybridize with or bind to cellular mRNA encodingthe polypeptide of interest to thereby inhibit expression, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell, e.g., a T cell or brain cell, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using vectors, e.g., genetherapy vectors, described below. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of interest can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gautier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Ribozymes are catalytic RNA molecules with ribonuclease activity thatare capable of cleaving a single-stranded nucleic acid, such as an mRNA,to which they have a complementary region, and can also be generatedusing standard techniques. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haseloff and Gerlach, 1988, Nature 334:585-591)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptide ofinterest can be designed based upon the nucleotide sequence of a cDNAencoding a cancer-associated variant of Mammalian Scratch. For example,a derivative of a Tetrahymena L-19 IVS RNA can be constructed in whichthe nucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, anmRNA encoding a polypeptide of interest can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak, 1993, Science 261:1411-1418.

Triple helical structures can also be generated using well knowntechniques. For example, expression of a polypeptide of interest can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the gene encoding the polypeptide (e.g., thepromoter and/or enhancer) to form triple helical structures that preventtranscription of the gene in target cells. See generally Helene, 1991,Anticancer Drug Des. 6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci.660:27-36; and Maher, 1992, Bioassays 14(12):807-15.

In various embodiments, nucleic acid compositions can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic &Medicinal Chemistry 4(1): 5-23). As used herein, the terms “peptidenucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics,in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup et al.,1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can, for example, be modified, e.g., to enhance their stability orcellular uptake, by attaching lipophilic or other helper groups to PNA,by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. For example, PNA-DNAchimeras can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAseH and DNA polymerases, to interact with the DNA portion while the PNAportion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup, 1996, supra, and Finn et al., 1996,Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can besynthesized on a support using standard phosphoramidite couplingchemistry and modified nucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., 1989,Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Petersen et al., 1995, Bioorganic Med.Chem. Lett. 5:1119-1124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652;International Publication No. WO 88/09810) or the blood-brain barrier(see, e.g., International Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., van der Krol et al., 1988, Bio/Techniques 6:958-976)or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).To this end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

Another aspect of the invention is a method to identify compounds thatare able to modulate the expression or activity of the cancer-associatedvariant of Mammalian Scratch, which can be used to prevent or treatcancer. In one embodiment of the invention, the method for identifying acompound for ability to prevent or treat cancer comprises the steps:

-   -   (a) contacting a cell expressing a cancer-associated variant of        Mammalian Scratch with a test compound; and    -   (b) determining the expression or function of the        cancer-associated variant of Mammalian Scratch; and    -   (c) comparing the expression or function of the        cancer-associated variant of Mammalian Scratch to a control,        wherein a decrease in expression or function of the        cancer-associated variant of Mammalian Scratch as compared to        the control is indicative of a compound useful to prevent or        treat cancer.

(D) Binding Proteins

Another aspect of the invention is a binding protein, preferably anantibody or antibody fragment, that binds to the isolated proteins ofthe invention. Such a binding protein can be generally referred toherein as “a binding protein of the invention”, or preferably “anantibody or antibody fragment of the invention”.

In one embodiment, the invention includes a binding protein that isspecific for a cancer-associated variant of Mammalian Scratch. In apreferred embodiment, the cancer-associated variant of Mammalian Scratchcomprises the amino acid sequence defined by SEQ ID NO:1 or a variantthereof or the amino acid sequence defined by SEQ ID NO:2 or a variantthereof. In another embodiment, the binding proteins bind to an isolatedprotein comprising the amino acid sequence defined by SEQ ID NO:1 or avariant thereof or the amino acid sequence defined by SEQ ID NO:2 or avariant thereof.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Furthermore,the antibody or antibody fragment can comprise all or a portion of akappa light chain constant region or a lambda light chain constantregion.

The isolated proteins of the invention may be used to prepare monoclonalor polyclonal antibodies. Conventional methods can be used to preparethe antibodies. For example, see Goding, J. W., Monoclonal Antibodies:Principles and Practice, 2^(nd) Ed., Academic Press, London, 1986.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, such as antigens or molecules on a cancer cell,may also be generated by screening expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria withcell surface components. For example, complete Fab fragments, VH regionsand FV regions can be expressed in bacteria using phage expressionlibraries (See for example Ward et al., Nature 341:544-546 (1989); Huseet al., Science 246:1275-1281 (1989); and McCafferty et al., Nature348:552-554 (1990)).

The invention also provides compositions comprising the binding proteinsof the invention, preferably antibodies and antibody fragments, with apharmaceutically acceptable excipient, carrier, buffer or stabilizer.

In addition, the binding proteins of the invention can be used in thediagnosis of cancer.

In a preferred embodiment, the binding proteins are antibodies orantibody fragments that bind to cancer-associated variants of MammalianScratch that is expressed on the surface of cancer cells, preferably anisolated protein comprising any one of the amino acid sequences of SEQID NOS: 1 or 2. In addition, cancer cells may be evaluated to determinetheir susceptibility to the treatment methods of the invention by, forexample, obtaining a sample of the cancer cells and determining theability of the sample to bind to the binding proteins of the invention,preferably antibodies or antibody fragments.

Accordingly, the present invention includes diagnostic methods, agents,and kits that can be used by themselves or prior to, during orsubsequent to the therapeutic method of the invention in order todetermine whether or not cancer cells are present that express theantigen and can bind to the binding proteins of the invention,preferably antibodies and antibody fragments.

In one embodiment, the invention provides a method of detecting ormonitoring cancer in a subject comprising the steps of

-   -   (1) contacting a test sample taken from said subject with a        binding protein that binds specifically to an antigen on the        cancer cell to produce a binding protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

In one embodiment, the antigen is a cancer-associated variant ofMammalian Scratch, preferably an isolated protein comprising any one ofthe amino acid sequences of SEQ ID NOS:1-2.

The invention further includes a kit for detecting or monitoring cancercomprising any one of the binding proteins of the invention that bindsto an antigen on the cancer cell and instructions for the use thereof.

For use in the diagnostic applications, the binding proteins of theinvention, preferably antibodies or antibody fragments, may be labeledwith a detectable marker such as a radio-opaque or radioisotope, such as³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) orchemiluminescent (chromophore) compound, such as fluoresceinisothiocyanate, rhodamine or luciferin; an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase; an imagingagent; or a metal ion. As described above, methods of attaching a labelto a binding protein, such as an antibody or antibody fragment, areknown in the art.

Another aspect of the invention is a method of detecting or monitoringcancer in a subject comprising the steps of

-   -   (1) measuring the amount of antibodies of the invention in a        test sample taken from said subject; and    -   (2) comparing the amount of antibodies of the invention in the        test sample to a control.

In one embodiment, the amount of antibodies of the invention is measuredby measuring the amount of antibodies of the invention in the testsample, for example by ELISA. In another embodiment, the amount ofantibodies of the invention is measured by measuring the expressionlevels of nucleic acids encoding the antibodies of the invention in thetest sample, for example by RT-PCR.

(E) Preparation of Proteins of the Invention

A person skilled in the art will appreciate that the proteins of theinvention, such as the novel cancer-associated antigen, the bindingproteins, preferably antibodies and antibody fragments, may be preparedin any of several ways, but is most preferably prepared usingrecombinant methods.

Accordingly, the nucleic acid molecules of the present invention may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the proteins of the invention. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses (e.g. replication defective retroviruses, adenovirusesand adeno-associated viruses), so long as the vector is compatible withthe host cell used. The expression vectors are “suitable fortransformation of a host cell”, which means that the expression vectorscontain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g. a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition,Cold Spring Harbor Laboratory Press, 2001), and other laboratorytextbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). In addition,the proteins of the invention may be expressed in prokaryotic cells,such as Escherichia coli (Zhang et al., Science 303(5656): 371-3(2004)). In addition, a Pseudomonas based expression system such asPseudomonas fluorescens can be used (US Patent Application PublicationNo. US 2005/0186666, Schneider, Jane C et al).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Protocols for the transformation of yeast and fungi are wellknown to those of ordinary skill in the art (see Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58(1987), which reviews the use of Agrobacterium rhizogenes vectors; seealso Zambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork (1984), which describes the use of expression vectors for plantcells, including, among others, PAPS2022, PAPS2023, and PAPS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Luckow, V. A., andSummers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect cellexpression systems suitable for expression of the recombinant proteinsof the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as mouse, rats, rabbits, sheep andpigs (Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964);Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesis inhomogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

N-terminal or C-terminal fusion proteins comprising the proteins of theinvention conjugated with other molecules, such as proteins may beprepared by fusing, through recombinant techniques. The resultant fusionproteins contain a protein of the invention fused to the selectedprotein or marker protein as described herein. The recombinant proteinof the invention may also be conjugated to other proteins by knowntechniques. For example, the proteins may be coupled usingheterobifunctional thiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins which may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Accordingly, the invention provides a recombinant expression vectorcomprising the nucleic acid sequences that encode the proteins of theinvention, such as the isolated proteins of the invention. Further, theinvention provides a host cell comprising the recombinant expressionvector of the invention.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Isolation and Identification of Cancer AssociatedScratch Experimental Design

Melanoma cell line (A-375), glioma cell lines (U118MG and U87MG), breastcancer cell line (MDA-MB 435S), pancreatic cell line, (PANC-1) andT-cell line (Daudi) were used in the study (Table 1). These cell lineswere selected based on the results of tumor cell line profiling by flowcytometry.

Growth and Maintenance of Tumor Cell Lines

The cell lines in the study were purchased from ATCC and cultured inaccordance with the guidelines and recommendations of ATCC. Cells wereharvested at 90% confluence with viability >90%.

Preliminary Characterization of the Antigen Binding to VB3-011

Preliminary characterization data was obtained from experiments designedto assess the feasibility of the gel-based approach by dot blot assays;and from experiments performed to determine the nature of the epitopeassociated with the antigens.

The data from these experiments classified the VB3-011 antigen as a“non-blottable” antigen with a glycan modification, i.e., the epitopeinvolved in binding to VB3-011 on the antigen was glycosylated.

VB3-011 Ag Enrichment and Purification

The preliminary data from the blottability study specified alectin-based purification method as the best antigen preparation methodfor VB3-011. Extensive experimentation revealed that the glycanmodification involved a soluble form of CS (chondroitin sulphate); twoof these (CSB and CSE) have limited tissue distribution. As such, theglycan modification could be attributable to CSA and to a lesser extenthyaluronic acid.

Chondroitin sulphate A (CSA) is made up of linear repeating unitscontaining D-galactosamine and D-glucuronic acid. The amino group ofgalactosamines in the basic unit of chondroitin sulfate A is acetylated,yielding N-acetyl-galactosamine; there is a sulfate group esterified tothe 4-position in N-acetyl-galactosamine (FIG. 1A) (Sugahara K et al.1988. J. Biol. Chem. Vol. 263:10168-10174; Sugahara K et al. 1991. Eur.J. Biochem. Vol. 202:805-811; Prydz K and Dalen K T. 2000. J. Cell Sci.Vol. 113:193-205). When these linear repeating units get cross-linked (α2-6) at branch points at C2 of the second and C6 of the first carbonchains, such that a single unit of glycan representing more than onelinear chains of CSA are present, except for the sulfation, it resemblesthe glycan, Neu5Ac (α 2→6) Gal(β 1→4) Glucuronate, recognized by HA(FIG. 1B).

Two or more CSA molecules when cross-linked together resemble theglycan—Neu5Ac (α 2→6) Gal (β 1→4) Glucuronate, recognized byHemagglutinin (HA), Azumi et al., (1991) showed that the activity of ahemagglutinin isolated from hemocytes of the ascidian, Halocynthiaroretzi was inhibited by heparin, chondroitin sulfate, andlipopolysaccharide (LPS), but not by mono- and disaccharides such asN-acetyl-galactosamine, galactose, and melibiose. The hemagglutininshowed binding ability to heparin, chondroitin sulfate and LPS, asdemonstrated by heparin-Sepharose chromatography and centrifugationexperiments, respectively (Ajit Varki et al eds. 1999. Essentials ofGlycobiology). Similarly, a Hemagglutinin from mycobacterium was shownto bind to heparan sulfate and Hemagglutinin from Hemophilius influenzaebinds to CSA with an additional α 2-6 linkage (Azumi K et al. A1991.Dev. Comp. Immunol. Vol. 15(1-2):9-16; Menozzi F D et al. 11996. J. Exp.Med., Vol. 184(3):993-1001). Heparan sulfate and Chondroitin sulfate Adiffer in C5 epimerization. Therefore, a new reagent that would enablelectin-based purification was generated as follows. Recombinant HA wasimmobilized to anti-HA antibody by coupling with Dimethylpimelimidate(DMP), such that when used as an IP agent, HA recognizes the CSAassociated with the antigen on the cell surface. Membrane preparationswere affinity purified with immobilized-HA, and the eluates subjected toSDS-PAGE and WB analysis, subsequently probed with VB3-011 antibody.

Lectin-Based Purification

Recombinant HA molecule that binds specifically to the glycan—Neu5Ac (α2→6) Gal (β 1→4) Glc, was made to bind to anti-HA antibody for 2 hoursat room temperature on the nutator, followed by binding of theHA-anti-HA complex to Protein-G-sepharose. This was followed by acentrifugation step to get rid of the unbound fraction. The immobilizedcomplex was then cross-linked using Dimethylpimelimidate (DMP) that isknown cross-link proteins present in close proximities. The excess orunused cross-linker and the unbound material were removed by a briefcentrifugation step. The non-specific amine groups that could havearisen as a by-product of the cross-linking step were neutralized withTriethanolamine for two hours at room temperature. The lectin-basedreagent thus created was washed thoroughly with PBS and stored with PBScontaining 0.05% NaN3 at 2-8° C. Apart from the HA-reagent,Con-A-agarose and WGA-agarose were also used as affinity purificationreagents to detect better antigen recovery.

A minimum of 500 μg membrane protein was used for the lectin-basedpurification. A pre-clearing step using protein-G sepharose alone wasthe first step in the purification of the antigen prior to the additionof the reagent. A total of 15-20 μL of the reagent was used as theprecipitating agent in the mixture. The antigen-lectin mixtures werenutated overnight at 4° C. using buffer conditions that mimickedphysiologic conditions. Care was taken to ensure that proteaseinhibitors were used in every step of the antigen isolation process.

Antigen-lectin complexes were centrifuged, washed with RIP-A lysisbuffer and eluted with 0.2 M glycine pH 2.5. Supernatants representingthe unbound fractions were stored to test the proteins that were notisolated by affinity purification. Lectin-based purifications werecarried out on two glioma cell lines (U118MG and U87MG), one melanomacell line (A-375), one epithelial cell line (MDA-MB-435S) and twonegative cell lines (Panc-1; and Daudi).

Gel-Based Analysis and Western Blotting

1D-PAGE:

The purified proteins were subjected to reducing conditions of samplepreparation and were subsequently analyzed by SDS-PAGE/Western Blotting.When reducing conditions were used, the isolated antigens were treatedwith sample buffer containing 1% β-mercaptoethanol at 65° C. for 15minutes. The resulting blots were probed with VB3-011 and correspondingsecondary antibodies conjugated to HRP, to visualize the purifiedproteins by chemiluminescence.

2D-PAGE:

The purified proteins were separated by two-dimensional gelelectrophoresis to resolve any protein stacking effect that may haveoccurred in the 1D-PAGE analysis. The 2D-gel electrophoresis resolvedproteins according to their isoelectric points (pI) in the firstdimension and on the basis of their molecular weights in the seconddimension. Proteins thus resolved were transferred to nitrocellulosemembranes, overnight, and processed as in the case of 1D-PAGE. Westernblots were probed with VB3-011 and reacting proteins visualized bychemiluminescence.

Peptide Extraction and Antigen ID

Peptide Extraction from in-Gel and in-Solution Tryptic Digests:

Tryptic digestions were performed with sequencing grade trypsin in a20-hour peptide extraction process finally resulting in the extractionof peptides that were analyzed on a QSTAR Pulsar-I (ESI-qTOF-MS/MS),equipped with a nanosource with a working flow rate of 20-50 nL/min. Thepeptides ionize and are detected as doubly, triply or quadruply chargedmolecules which are then refined to their respective masses. De-novosequencing of the identified proteins was also performed wheneverpossible. Peptides were extracted from both positive and negative celllines to ensure it was the right antigen. Peptide masses extracted fromthe mass spectra were used directly to identify the antigen according tothe MOWSE scores obtained on protein databases that are accessiblethrough the MASCOT search engine. Peptides were extracted both from gelslices and in-solution (U118MG, U87MG, A-375, 435S) and subjected themto MS analysis.

Results HA Reagent Immobilization

Recombinant HA molecule is not an antibody and therefore does not bindto protein-G-sepharose directly as an immobilizing partner. In order tomake it possible for this molecule to be functional in antigenpurification processes, HA was bound to anti-HA antibody that would bindspecifically to HA, the molecule was immobilized usingprotein-G-sepharose in a sequential manner. This would not onlyimmobilize the complex but would block any non-specific interaction thatcould arise from the presence of the anti-HA, as shown schematically inFIG. 2. The immobilized HA-anti-HA complex was thereafter stabilizedusing Dimethyl pimelimidate, a cross-linking agent that maintained theproximities of the various reactants. The final complex generated a fewreactive amines in the process, other than the reactive binding site onthe HA molecule. These reactive groups were blocked permanently using 1Mtriethanolamine, thus ensuring the maximal exposure of the reactive siteon the HA molecule.

Lectin-Purification

All purification reactions were performed with pre-cleared proteins.Longer incubation times were used to minimize non-specificity andenhance the stability of cognate antigen-antibody complexes. Six celllines (A-375, U118MG, U87MG, MDA-MB-435S, Panc-1 and Daudi) were used inthe study. Reducing conditions for sample preparations were employedprior to the resolution of the antigens isolated on SDS-PAGE. TheWestern blots were probed with VB3-011 to ensure that the antigenpurified is the cognate binding partner for VB3-011.

1D-PAGE/Western Analysis

When HA reagent was used, only one specific band was detected afterseparation on a 1D-PAGE at ˜50 kDa under reducing conditions (FIG. 3A)in antigen-positive cell line (A-375), that was absent in the negativecell line (Panc-1). Non-specific interactions were observed with Con-Aand WGA lectins indicating that the glycan present on the VB3-011antigen was the one recognized by HA. Glioma cell line (U118MG andU87MG) also showed the presence of a single band at ˜50 kDa whenpurified using the HA reagent (FIG. 3B). When samples were allowed tosit at room temperature for 1 hour prior to their separation onSDS-PAGE, a predominant band at ˜36 kDa and a faint 50 kDa band wereobserved in antigen-positive cell line (A-375, U118MG and U87MG) (FIG.4).

2D-PAGE Analysis

In order to determine isoelectric points (pI) and assess the possibilityof protein stacking in the 1D-PAGE analysis, the antigens purified by HAwere separated on two-dimensional polyacrylamide gel electrophoresis(2D-PAGE), where the separation in the first dimension is on the basisof pI and the second dimension on the basis of molecular weight. Thegels were then transferred to nitrocellulose membranes and subjected tostandard Western blotting processing. Since the amounts required for thedetection of proteins on a 2D gel is ˜4 times higher than therequirement for a 1D gel, purified antigens from 4 separate reactionswere pooled together for one 2D-PAGE analysis. Two separate gels wereprocessed simultaneously for Western blot analysis to ensure that theproteins detected on the Coomassie stained gels are the same as thoseobserved in the Western blots. The 2D Western blots were probed withVB3-011 and detected by ECL (chemiluniescence). As can be seen in FIG.5, one single spot was detected at ˜36 kDa/pI=9.7±0.2.

Peptide Extraction and Protein Analysis

A-375, U 87MG and U118 MG membranes were used to purify antigen(s) thatbind specifically to VB3-011. A ˜50 kDa band was observed in all threecell lines as shown in FIGS. 3A and 3B. The protein bands were excisedfrom the coomassie stained gels and used in-gel digestion to extractpeptides for MS analysis.

Proteins from 1D-gel band and 2D-spots were digested with trypsin torelease them from the gel and analyzed on a reverse-phase LC-MS/MSsystem. The identities of the proteins were revealed by databaseanalysis using bioinformatic tools. Raw data included peptides obtainedas listed in the TOF-MS spectra, MS/MS fragmentation data, and a list ofsuggested proteins including contaminants that do not match the pI orthe molecular weight of the protein isolated. To obtain the analysisMS/MS spectra were submitted directly to Mascot search engines availableat www.Matrixscience.com.

Mass Spectral Analysis

Peptide analysis was done in two ways:

-   -   All the peptides recovered and reconstructed to their right        masses were used directly in a peptide mass fingerprinting step        to obtain an ID for the protein.    -   Peptides that were abundant and well ionized were chosen for        further MS/MS ion fragmentation, wherein, the ‘y’ and ‘b’ ions        were used to deduce their primary structure. These sequences        were then searched for homologies in the protein database for        protein ID.

Peptides ionize and are detected as doubly, triply or quadruply chargedmolecules, on a LC-MS/MS system as opposed to detection as singlycharged on Matrix assisted ionization such as in MALDI. Differentiallycharged peptides were thereafter refined to their respective masses, inthe mass reconstruction step. These peptide masses were then directlyanalyzed by a matrix science based mascot search engine for antigen ID.Peptide masses extracted from the mass spectra were used directly toidentify the antigen according to the MOWSE scores obtained on proteindatabases that are accessible through search engines such as MASCOT,SEQUEST, and Prospector. QSTAR-pulsar-I was used and selected for allprotein identities, because it includes the most recent protein databaseadditions from Pepsea is compatible with MASCOT.

Analysis of 2D Spot

Protein spot excised from the 2D-gel identified Scratch. The pI and themolecular weight clearly matched Mammalian Scratch. A total of 37%sequence coverage with 15 matching peptides, each peptide showing 100%homology to the original protein was recovered (See FIG. 6).

Analysis of the 50 kDa Band Purified from the Glioma and Melanoma CellLines

The data obtained from the mass spectra of all three cell lines, (U87MG,U118MG and A375) point towards Mammalian Scratch as the antigen thatbinds to VB3-011. Of all the cell lines screened, glioma cell lines(U87MG and U118MG) showed the highest scoring identities. A-375, amelanoma cell line also showed an over-expression of the antigen. Apartfrom the above mentioned cell lines, epithelial cell lines such asMDA-MB-435S, PC-3, A-549 and CFPAC-1 were also screened in the samemanner, but except for MDA-MB-435S, which showed the presence of atruncated version of Scratch, i.e., 17.823 kDa protein gi|15928387, with100% homology to sequences 158-366 of the original scratch molecule. SeeFIG. 7 (SEQ ID NO:4). The membrane preparations from each of these celllines were used to affinity purify the VB3-011 antigen using theHA-reagent. Rest of the epithelial cell lines showed no detectableproteins.

TOF-MS scans were obtained both on a manual mode and an IDA mode torecover the maximum number of peptides for a significant ID. See FIGS.8-10.

The list of peptides recovered and their mapped positions to thesequence from Mammalian Scratch are as given in FIG. 11 (SEQ ID NO:1)and Table 2 (SEQ ID NOS:2 and 7 to 24). All peptides represented wereobtained by de novo sequencing.

MS/MS Fragmentation of Peptide 2402.1206 and 2134.9614

A discrete nanospray head installed on a nanosource was used for thepurpose. The collision energy was 48V, curtain gas and CAD gas weremaintained at 25 and 6, respectively, and the sample allowed to cyclefor 1.667 minutes (100 cycles) to obtain stable mass ion fragmentation.MS/MS fragmentation of two of the peptides (2402.978172-802.00000, 3+;2134.985448-1068.500000, 2+) gave rise to the fragment ions shown inFIGS. 14 and 15. While one of the peptides, ‘PELATAAGGYINGDAAVSEGYAADAF’(SEQ ID NO:7) from peptide mass 2402.97812, mapped 100% to a sequencefrom Scratch, peptide, RFLAAFLAAAGPFGFALGPSSV (SEQ ID NO:2), frompeptide mass 2134.985448, showed 100% homology in the flanking sequencesbut not with the sequence in the middle, indicating an identification ofa novel sequence. The presence of this sequence is responsible for theonly transmembrane domain available on the protein. Mammalian Scratchsequence available in the database is a result of conceptual translationand does not have any transmembrane domains in the sequence. The proteinsequence recovered shows 67% homology to the Mammalian Scratch proteinavailable in the database and indicative of being present on the cellsurface due to the presence of a transmembrane domain. Rest of thepeptides derived from the spectra clearly matched the sequences fromMammalian Scratch, and therefore were pulled down as major hits. The ionfragmentation data further confirm the identity of a novel form ofScratch as the cognate antigen for VB3-011.

FIGS. 12 and 13 identify Mammalian Scratch as the antigen.

Discussion

VB3-011, an IgG MAb, was generated from peripheral blood lymphocytes(PBL) isolated from a patient diagnosed with a grade II astrocytoma,using Hybridomics™ and ImmunoMine™ Viventia's proprietary platformtechnologies (See WO97/044461). The antibody exhibits reactivity to ahost of other cell lines each of which is representative of differentcancer indications. Despite this demonstration of broad tumor-cell typereactivity, VB3-011 shows limited binding to normal tissue. VB3-011antigen was classified as a “non-blottable” antigen with a glycanmodification, attributable to CSA.

Since CSA molecules are characterized by (1-4) GlcNAc/Glucuronatestructures they also resemble the lectin—Neu5Ac (α2→6)Gal(β1→4)Glucuronate, recognized by Hemagglutinin (HA). A new reagentthat would enable lectin-based purification was generated using.recombinant HA was immobilized to anti-HA antibody as an purificationagent. Membrane preparations were affinity purified with immobilized-HA,and the eluates subjected to SDS-PAGE and WB analysis, subsequentlyprobed with VB3-011 antibody. VB3-011 detected a ˜50 kDa protein on1D-PAGE that further resolved into a ˜36 kDa band on 2D-PAGE analysis.LC-MS/MS analysis of the 1D and 2D spots identified Mammalian Scratch asthe antigen with molecular weight 36 kDa (of ˜50 kDa observed by WBanalysis of 1D-PAGE), thus attributing the rest to the presence of theglycan, 4-sulfated, Neu5Ac (α2→6) Gal(β1→4)Glucuronate. The detection ofa 36 kDa spot on 2D-PAGE matched the molecular weight and isoelectricpoint [(pI), i.e., 9.7±0.2] characteristic of Mammalian Scratch.

The protein sequences recovered by denovo sequencing from MS/MS fragmention analyses, resulted in 67% coverage with 16 out of 17 peptidesshowing 100% homology to the Mammalian Scratch sequence found in thedatabase (gi|13775236). One peptide, RFLAAFLAAAGPFGFALGPSSV (SEQ IDNO:2), from peptide mass 2134.985448, showed 100% homology in theflanking sequences but not with the sequence in the middle, indicatingan identification of a novel sequence. The presence of this sequence isresponsible for the only transmembrane domain available on the proteinand places Scratch on the cell-surface as opposed to the cytosol. Thisis the first report depicting Mammalian Scratch as a cell-surface tumorantigen.

Example 2 Tumor Associated Expression of Scratch

An antibody specific for Mammalian Scratch was tested for tumorspecificity using HD formalin fixed TMA's. See Table 3 for normaltissues and Table 4 for tumor specific membrane binding. There was nodetection of the Scratch antigen on the membrane of normal tissue.However, strongly positive membrane staining was found on a variety oftumor tissues.

Example 3 Localization of Scratch as Cancer Diagnostic

Aberrant Localization of the Scratch Protein as an Indicator of Cancer:

Wild type Scratch protein has a limited expression pattern within thenucleus of cells as described by Nakakura et al, 2001. However,expression in the case of the tumor tissue types and cancer cell typeshas been established on the membrane and within the cytoplasm of thecells by the inventors. Using techniques known in the art such as flowcytometery, immunohistochemistry, western blotting of membrane fractionsof cells aberrant expression of the Scratch protein and variants thereofcan be established on the membrane and within the cytoplasm of cancercells. This change in localization can be used as a diagnosticindicative of cancer.

Membrane expression of variant Scratch proteins has been established byboth flow cytometery and western blotting of membrane fractions fromcancer cell types such as U-87Mg, A375, MDA-MB-435S, U118-MG. This isshown in Table 1, and FIGS. 3, 4 and 15.

Example 4 Detection of Variant mRNA as Indication of Cancer

RT-PCR Methodology for Sensitive Detection of Variant mRNA of MammalianScratch Containing Transmembrane Domain:

Messenger RNA will be isolated from different types of tumor cells andfirst strand complement DNA (cDNA) will be synthesized using the reversetranscriptase enzyme and an oligo dT primer. The cDNA will then be usedto test for the expression of the wild type Scratch mRNA and possiblevariants and specifically the transmembrane mutant by PCR using thefollowing primers:

5′ Primer 1: for wt and variant (corresponding to nucleotides 51 to 82)(SEQ ID NO: 26) 5′-GCC GAC CTG GAG AGC GCC TAC GGA CGC GCC 5′Primer 2: for transmembrane variant(corresponding to nucleotides 76 to 105) (SEQ ID NO: 27)5′-CGC GCC CGC TTX1 TTX2 GCX3 GCX3 TTX1 TTX2 GCX3Where X1 is T or C, X2 is A or G and X3 is A, G, C, or T 3′Primer: (corresponding to nucleotides 183 to 210) (SEQ ID NO: 28)5′-TGC GTA CAT GGG CTC CGG CGA CGG CCCThe PCR reaction included a 50 μL reaction volume containing:

10X PCR buffer 5 μL 2 mM dNTPs 5 μL Primer 5′ 20 pmol Primer 3′ 20 pmolTaq DNA Polymerase 2.5 U DNA template 50 ng

The cycling conditions for PCR were: 94° C. for 1 min., 62° C. for 1min., and 72° C. for 30 sec., for a total of 30 cycles followed by afinal extension of 10 min. at 72° C.

Electrophoresis on a 1% agarose gel will demonstrate that the band ofinterest of 159 bp is present in reactions using primer 1 and 140 bp inreactions using primer 2 if the transmembrane mutant is present.

The sequence analysis of the wild type Mammalian Scratch revealed a KpnIrestriction site (position 118) that is not present in the variant.Therefore, to test if the variant form is expressed in tumor cells, theamplified PCR product will be digested with the KpnI restriction enzymefollowed electrophoresis on a 1.5% agarose gel. If the tumor cellsexpress the wild type Mammalian Scratch, then two fragments of 67 and 92bp will be detected under UV lamp. In contrast, if the tumor cellsexpress a variant of Scratch, lacking the KpnI site then the size of thePCR fragment will be identical to the undigested control. Using theprimers specific to the transmembrane region of the variant MammalianScratch (primer #2) a PCR fragment will only be found in samplescontaining the variant with the transmembrane domain, therebyidentifying the specific variant.

Example 5 Detection of Genomic DNA Sequence as an Indication of Cancer

The gene coding for the human Mammalian Scratch protein has been locatedto chromosome 8 q24.3 and consists of 2 exons. The gene sequence for thecancer associated membrane bound variant of the Scratch can be easilydetermined using gene sequencing techniques known in the art such asexon-specific PCR amplification, or direct DNA sequencing initiatingfrom primers to the known sequence.

Once the sequence of the mutated gene is known diagnostic tests based onits detection can be used to evaluate patients. DNA chip arrays can becreated by attaching oligonucleotides corresponding to the sense andantisense sequences of both wild type and the mutated gene Genomic DNAcan be isolated from the peripheral whole blood or from tumor tissuesThe gene of interest is then amplified using PCR with primerscorresponding both to the wild type sequence and to the expectedmutations and labeled with an appropriate probe (usually fluorescent).The DNA is then hybridized to the oligonucleotides on the chip and thepattern of fluorescence determined with a fluorescent reader. Bycomparing the pattern of fluorescence to a map of the known locations ofthe oligonucleotides sequences the sequence of the patients gene withcan be established as either wild type or mutant. (Cooper et al 2004)

Arrays for common mutations in the p53 gene (Affymetrix) among othersare already commercially available and custom array services are alsoavailable

Example 6 Variant Cancer Associated Scratch as a Target for Immunotoxins

VB6-011 is a immunoconjugate of modified bouganin conjugate with anantibody that specifically recognizes Mammalian Scratch protein on thetumor cell surface. Treatment of cells expressing variant Scratchcontaining a transmembrane domain on the cell surface results inspecific uptake of the immunoconjugate and subsequent cell death.

Cytotoxicity of VB6-011 Proteins

The cytotoxicity of VB6-011 was measured by an MTS assay. Briefly,antigen-positive and antigen-negative cells were seeded at 1000 cellsper well and incubated at 37° C. for 3 hours. Subsequently, varyingconcentrations of VB6-011 and de-bouganin were added to the cells andafter 5 days, the cell viability determined.

The negative and positive-antigen cell lines were incubated withdifferent concentrations of VB6-011 from 1 nM to 1 mM. After 5 daysincubation, the calculated IC₅₀ of VB6-011 was 350 nM. (FIG. 18) (Table5) In contrast, no IC₅₀ could be determined with the antigen negativecell lines.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Cell line MF* A375 11.5  U118MG 6.1 U87MG 4.6 MDA-MB-435S 4.6PANC-1 2.1 DAUDI 1.1

-   -   Table 1: Increase in median fluorescence for VB3-011 over an        isotype-matched control for each cell line used in the study.

TABLE 2  Peptide Start End mass Description (Peptide sequence) 28 50A.RFLAAFLAAAGPFGFALGPSSV.Y (SEQ ID NO: 2) 92 117R.PELATAAGGYINGDAAVSEGYAADAF.F (SEQ ID NO: 7) 4 8 592.7360R.SFLVK.K (SEQ ID NO: 8) 12 26 1601.6900K.LDAFSSADLESAYGR.A (SEQ ID NO: 9) 62 74 1345.5360K.GPSPEPMYAAAVR.G (SEQ ID NO: 10) 75 123 4719.1140R.GELGPAAAGSAPPPTPRPELATAAGGYINGDAAVSEGYAADAFFITDGR.S (SEQ ID NO: 11)128 158 2457.4690 K.ASNAGSAAAPSTASAAAPDGDAGGGGGAGGR.S (SEQ ID NO: 12)159 167 786.8430 R.SLGSGPGGR.G (SEQ ID NO: 13) 172 179 731.7640R.AGAGTEAR.A (SEQ ID NO: 14) 180 190 840.8940R.AGPGAAGAGGR.H (SEQ ID NO: 15) 199 208 1099.1660K.TYATSSNLSR.H (SEQ ID NO: 16) 215 222 888.9760R.SLDSQLAR.R (SEQ ID NO: 17) 230 247 2085.5280K.VYVSMPAMAMHLLTHDLR.H (SEQ ID NO: 18) 256 268 1598.8890K.AFSRPWLLQGHMR.S (SEQ ID NO: 19) 284 288 578.6260K.AFADR.S (SEQ ID NO: 20) 293 302 1157.3120R.AHMQTHSAFK.H (SEQ ID NO: 21) 312 316 564.6820K.SFALK.S (SEQ ID NO: 22) 317 321 623.7070 K.SYLNK.H (SEQ ID NO: 23) 330348 1642.8320 K.GGAGGPAAPAPPQLSPVQA. (SEQ ID NO: 24)

-   -   Table 2: List of peptides along with their respective calculated        masses obtained after the reconstruction step is as given in the        above table.

TABLE 3 TMA assessment of Antigen Binding Fragment Reactivity withnormal tissues by IHC Tissue Location of Staining Array # Element Score*Comments Adrenal Cytoplasm 1 Cortex + Not Scored None 1 & 2 Medulla 0Cytoplasm 2 Cortex 1 Bone Marrow None 1 & 2 Not Applicable 0 Brain None1 Neurons 0 None 2 Neurons Trace Cytoplasm 1 Astrocytes 1 Cytoplasm 2Astrocytes Trace Breast None 1 & 2 Not Applicable 0 Cartilage None 1 & 2Not Applicable 0 Colon None 1 & 2 Not Applicable 0 Heart None 1 & 2 NotApplicable 0 Kidney None 1 & 2 Glomeruli 0 Cytoplasm 1 & 2Tubules(Proximal & Distal) 1 Liver Cytoplasm 1 & 2 Hepatocytes 1Cytoplasm 1 Bile ducts 0 to 1+ None 2 Bile ducts 0 Lung None 1 & 2 NotApplicable 0 Ovary None 1 & 2 Not Applicable 0 Pancreas Cytoplasm 2Acini 2 Scattered cells Cytoplasm 1 Acini Trace to 3+ None 1 & 2 Ductalcells 0 None 2 Islet cells 0 Cytoplasm 1 Islet cells Trace PeripheralNerve None 1 & 2 Not Applicable 0 Prostate None 1 & 2 Not Applicable 0Salivary Gland Cytoplasm 1 & 2 Ductal cells 1 None 1 & 2 Acini 0Skeletal Muscle None 1 & 2 Not Applicable 0 Skin None 1 & 2 NotApplicable 0 Spleen None 1 & 2 Not Applicable 0 Stomach None 1 & 2 NotApplicable 0 Testis Cytoplasm 1 Germ cells Trace None 2 Leydig cells 0None 2 Germ cells 0 Cytoplasm 1 Leydig cells 1 Thyroid None 1 & 2 NotApplicable 0 *Scoring was evaluated on a 0-4+ scale, with 0 = nostaining and trace being less than 1+ but greater than 0. Grades 1+ to4+ represent increased intensity of staining, with 4+ being strong, darkbrown staining.

TABLE 4 Tumor TMA analysis for Antigen Binding Fragment Membrane Tissuestaining Score* Comments Lymphoma  7/10 1 to 3+ Strong membrane stainingBreast Carcinoma 27/31 1 to 3+ Strong membrane staining Colon Carcinoma23/26 1 to 3+ Prominent membrane reactivity Melanoma 13/14 1 to 3+Prominent membrane reactivity Prostate Carcinoma 17/20 1 to 2+ Majoritywere strongly positive Cervix Squamous 22/24 1 to 2+ Majority—stronglypositive Cell Carcinoma Cervix 9/9 1 to 2+ Majority—strongly positiveAdenocarcinoma Kaposi Sarcoma 7/8 1 to 2+ Majority were stronglypositive *Scoring was evaluated on a 0-4+ scale, with 0 = no stainingand trace being less than 1+ but greater than 0. Grades 1+ to 3+represent increased intensity of staining, with 4+ being strong, darkbrown staining. nd: not determined.

TABLE 5 Biological characterization of VB6-011 VB6 Saturation IgGconcentration Affinity (M) conc. (μg/mL) (μg/mL)* IC₅₀ (nM) VB6-011 2 ·10⁻⁶ 250 180 350 ND: not determined. *Concentration of IgG that inhibits50% of the VB6 binding.

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1-31. (canceled)
 32. An isolated antibody or an antibody fragment thatbinds to a protein comprising an amino acid sequence of SEQ ID NO: 1, ora variant thereof.
 33. The isolated antibody or the antibody fragment ofclaim 32, wherein the protein is a variant of mammalian Scratch.
 34. Theantibody fragment of claim 32, wherein the antibody fragment is selectedfrom the group consisting of Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, minibodies, diabodies, multimers thereof, and bispecificantibody fragments.
 35. An isolated antibody or an antibody fragmentthat binds to a protein comprising an amino acid sequence of SEQ ID NO:2, or a variant thereof.
 36. The antibody fragment of claim 35, whereinthe antibody fragment is selected from the group consisting of Fab,Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies,multimers thereof, and bispecific antibody fragments.
 37. A compositioncomprising an isolated antibody or an antibody fragment of claim 32 witha pharmaceutically acceptable excipient, carrier, buffer, or stabilizer.38. A composition comprising an isolated antibody or an antibodyfragment of claim 35 with a pharmaceutically acceptable excipient,carrier, buffer, or stabilizer.
 39. An immunoconjugate comprising anantibody or an antibody fragment that binds to a protein comprising anamino acid sequence of SEQ ID NO: 1, attached to an effector molecule.40. The immunoconjugate of claim 39, wherein the antibody fragment isselected from the group consisting of Fab, Fab′, F(ab′)2, scFv, dsFv,ds-scFv, dimers, minibodies, diabodies, multimers thereof, andbispecific antibody fragments.
 41. The immunoconjugate of claim 39,wherein the antibody or the antibody fragment is humanized.
 42. Theimmunoconjugate of claim 39, wherein the effector molecule is selectedfrom the group consisting of a toxin, an enzyme conjugate, a labeledderivative, a fluorophore, a chromophore, an imaging agent, and a metalion.
 43. The immunoconjugate of claim 39, wherein the effector moleculeis a modified bouganin.
 44. The immunoconjugate of claim 39, wherein theimmunoconjugate is VB6-011.
 45. A composition comprising animmunoconjugate of claim 39 and a pharmaceutically acceptable excipient,carrier, buffer, or stabilizer.
 46. An immunoconjugate comprising anantibody or an antibody fragment that binds to a protein comprising anamino acid sequence of SEQ ID NO: 2, attached to an effector molecule.47. The immunoconjugate of claim 46, wherein the antibody fragment isselected from the group consisting of Fab, Fab′, F(ab′)2, scFv, dsFv,ds-scFv, dimers, minibodies, diabodies, multimers thereof, andbispecific antibody fragments.
 48. The immunoconjugate of claim 46,wherein the antibody or the antibody fragment is humanized.
 49. Theimmunoconjugate of claim 46, wherein the effector molecule is selectedfrom the group consisting of a toxin, an enzyme conjugate, a labeledderivative, a fluorophore, a chromophore, an imaging agent, or a metalion.
 50. The immunoconjugate of claim 46, wherein the effector moleculeis a modified bouganin.
 51. A composition comprising an immunoconjugateof claim 46 and a pharmaceutically acceptable excipient, carrier,buffer, or stabilizer.
 52. A kit for diagnosing cancer comprising anisolated antibody or an antibody fragment that binds to a proteincomprising an amino acid sequence of SEQ ID NO: 1 and instructions forthe use thereof to diagnose cancer.
 53. A kit for treating or preventingcancer comprising an effective amount of the immunoconjugate of claim39, and directions for the use thereof to treat or prevent cancer.
 54. Akit for diagnosing cancer comprising an isolated antibody or an antibodyfragment that binds to a protein comprising an amino acid sequence ofSEQ ID NO: 2 and instructions for the use thereof to diagnose cancer.55. A kit for treating or preventing cancer comprising an effectiveamount of the immunoconjugate of claim 46, and directions for the usethereof to treat or prevent cancer.